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WO1999067290A1 - Novel hemopoietin receptor proteins - Google Patents

Novel hemopoietin receptor proteins Download PDF

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Publication number
WO1999067290A1
WO1999067290A1 PCT/JP1999/003351 JP9903351W WO9967290A1 WO 1999067290 A1 WO1999067290 A1 WO 1999067290A1 JP 9903351 W JP9903351 W JP 9903351W WO 9967290 A1 WO9967290 A1 WO 9967290A1
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Prior art keywords
amino acid
protein
seq
dna
acid sequence
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PCT/JP1999/003351
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French (fr)
Japanese (ja)
Inventor
Hitoshi Nomura
Masatsugu Maeda
Original Assignee
Chugai Research Institute For Molecular Medicine, Inc.
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Publication date
Application filed by Chugai Research Institute For Molecular Medicine, Inc. filed Critical Chugai Research Institute For Molecular Medicine, Inc.
Priority to EP99926775A priority Critical patent/EP1088831A4/en
Priority to CA002330547A priority patent/CA2330547A1/en
Priority to AU43922/99A priority patent/AU759689B2/en
Publication of WO1999067290A1 publication Critical patent/WO1999067290A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons

Definitions

  • the present invention relates to a novel hemopoietin receptor protein, a gene encoding the same, a method for producing the same, and a use thereof.
  • cytokines are known as humoral factors involved in the proliferation and differentiation of various cells or the activation of differentiated and mature cells and the cell death.
  • Each of these site receptors has specific receptors, and these receptors have been classified into several families based on their structural similarities (Hilton DJ, in "Guidebook to Cytokines and Their Receptors "eaited by Nicola NA (A Sambrook & Tooze Publication at Oxford University Press), 1994, p8-16).
  • cytokine family include tyrosine kinase receptor, hemopoietin receptor, tumor necrosis factor (TNF) receptor, and transforming growth factor /? (TGF?) Receptor.
  • TGF tumor necrosis factor
  • TGF tumor necrosis factor
  • TGF tumor necrosis factor
  • TGF tumor necrosis factor
  • TGF transforming growth factor /?
  • hemopoietins are regarded as hormone-like factors.
  • receptors for growth hormone, prolactin, or lebutin which are typical peptide sex hormones, also belong to the hemopoietin receptor family. From the above-mentioned hormone-like systemic regulation mode, application of these hemopoetins to treatment of various diseases is expected.
  • site force receptors have structural similarities among family members. Numerous attempts have been made to discover new receptors using this similarity, and particularly for tyrosine kinase receptors, many receptors have already been cloned using highly conserved sequences in their catalytic sites. (Matthews W. et al., Cell, 1991, 65 (7) pll43-52). In contrast, the hemopoietin receptor does not have an enzymatically active domain such as tyrosine kinase in its cytoplasmic region, and its signal transduction is associated with another tyrosine kinase protein that exists free in the cytoplasm. It is known to take place through a meeting.
  • Binding sites on receptors for these cytoplasmic tyrosine kinases Although they are conserved between members of the family, their homology is not very high (Murakami M. et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 11349-11353).
  • the sequences that best characterize these hemopoietin receptors are rather present in the extracellular region, and in particular, the motif consisting of 5 amino acids of Trp-Ser-Xaa-Trp-Ser (Xaa is any amino acid) is almost all It is conserved in the receptor, and it is expected that a new receptor will be obtained by searching for a new family member using this sequence.
  • IL-11 receptor Robot, L. et al., J. Biol. Chem. 271 (23), 1996, 13754-13761
  • lebutin receptor Gainsford T. et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (25) pl4564-8)
  • IL-13 receptor Hilton. DJ et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (1) p497 -501
  • the present invention provides a novel hemopoietin receptor protein and a DNA encoding the same.
  • the present invention also provides a vector into which the DNA has been inserted, a transformant retaining the DNA, and a method for producing a recombinant protein using the transformant.
  • the present invention further provides a method for screening for a compound that binds to the protein.
  • the present inventors have previously proposed a novel receptor by a method such as plaque hybridization or RT-PCR using an oligonucleotide coding for the Trp-Ser-Xaa-Trp-Ser motif as a probe.
  • clones derived from the human genome by converting the nucleotide sequence around the probe sequence to an amino acid sequence and comparing it with the amino acid sequence of a known hemopoietin receptor, a human gene thought to encode a member of the hemopoietin receptor familly was selected. Different.
  • hemopoietin receptor genes Two clones considered to be the hemopoietin receptor genes were identified. One of them is a known GM-CSF vector receptor gene (chromosome 22 from 22ql 2.3-13.2 region) and the other (chromosome 16 from 16pl2 region BAC clone A C002303) is a novel hemopoietin receptor. It was presumed to encode the body, and this human gene was named "Vol. 8".
  • a cDNA that is thought to encode NR8 was found in a human fetal liver cell cDNA library by RT-PCR using a specific primer designed based on the obtained nucleotide sequence. . Furthermore, by performing the 5'-RACE method and the 3'-RACE method using this cDNA library as a type I, a full-length cDNA encoding a 361 amino acid transmembrane receptor, NR8, was finally obtained. Obtained.
  • cysteine residues conserved among other family members in the extracellular region, proline-rich motifs, and the boxl motif thought to be involved in signal transduction in the intracellular region are well conserved. And was considered a typical hemopoietin receptor.
  • the present inventors tried to isolate a mouse gene corresponding to the NR8 gene.
  • cross-species crossover PCR cloning is performed using a mouse brain cDNA library as a type I, using an oligonucleotide primer designed within the cDNA sequence of human NR8.
  • the partial nucleotide sequence was isolated.
  • oligonucleotide primers are designed based on the obtained partial sequences, and the 5, -RACE method and the 3'-RACE method using the primers are performed to obtain the entire length of the mouse homologous gene corresponding to NR8. 0RF was successfully isolated.
  • the present invention relates to (1) a protein consisting of the amino acid sequence from amino acid Me at position 1 to amino acid Ser at position 361 shown in SEQ ID NO: 1, or one or more amino acid sequences in the protein; Consisting of an amino acid sequence modified by the deletion, addition and / or substitution of another amino acid of the amino acid sequence, from the amino acid sequence from amino acid Met at position 1 to amino acid Ser at position 361 shown in SEQ ID NO: 1.
  • a protein consisting of the amino acid sequence from amino acid Me at position 1 to amino acid Ser at position 361 shown in SEQ ID NO: 1, or one or more amino acid sequences in the protein; Consisting of an amino acid sequence modified by the deletion, addition and / or substitution of another amino acid of the amino acid sequence, from the amino acid sequence from amino acid Met at position 1 to amino acid Ser at position 361 shown in SEQ ID NO: 1.
  • the present invention also relates to (2) a protein consisting of the amino acid sequence from amino acid 1 at position 1 to amino acid Leu at position 144 shown in SEQ ID NO: 3, or one or more amino acid sequences in the amino acid sequence in the protein. Deletions, additions and / or other amino acids It provides a protein consisting of an amino acid sequence modified by substitution with an amino acid and functionally equivalent to a protein consisting of the amino acid sequence from the first amino acid Met to the 144th amino acid Leu shown in SEQ ID NO: 3. .
  • the present invention also relates to (3) a protein consisting of the amino acid sequence from amino acid 1 at position 1 to amino acid Ser at position 237 shown in SEQ ID NO: 5, or one or more amino acid sequences in the amino acid sequence in the protein. Consisting of an amino acid sequence modified by deletion, addition and / or substitution of another amino acid, comprising the amino acid sequence from amino acid 1 at position 1 to amino acid Ser at position 237 shown in SEQ ID NO: 5.
  • the present invention also relates to (4) a protein consisting of the amino acid sequence from amino acid 1 at position 1 to amino acid Ser at position 538 shown in SEQ ID NO: 7, or one or more amino acid sequences in the protein. Consisting of an amino acid sequence modified by deletion, addition and / or substitution of another amino acid, and comprising an amino acid sequence from the 1st amino acid Met shown in SEQ ID NO: 7 to the 538th amino acid Ser.
  • the present invention also relates to (5) a protein consisting of the amino acid sequence from amino acid Me at position 1 to amino acid Leu at position 144 shown in SEQ ID NO: 19, or one or more amino acid sequences in the amino acid sequence in the protein. Consisting of an amino acid sequence modified by deletion, addition, and / or substitution of another amino acid, the amino acids from the 1st amino acid Met shown in SEQ ID NO: 19 to the 144th amino acid Leu It provides a protein functionally equivalent to the protein consisting of the sequence.
  • the present invention also relates to (6) a protein consisting of an amino acid sequence from amino acid Me at position 1 to amino acid Ser at position 538 shown in SEQ ID NO: 21 or one or more amino acid sequences in the amino acid sequence in the protein. Consisting of an amino acid sequence modified by deletion, addition and / or substitution of another amino acid, the amino acids from amino acid Met at position 1 shown in SEQ ID NO: 21 to amino acid Ser at position 538 Arrangement Provides a protein functionally equivalent to the protein in the sequence.
  • the present invention also provides (7) a protein encoded by a DNA that hybridizes with the DNA consisting of the nucleotide sequence of SEQ ID NO: 2, wherein the amino acid at position 1 to the amino acid at position 361 to the amino acid at position 361 shown in SEQ ID NO: 1 Provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to Ser.
  • the present invention also provides (8) a protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 4, wherein the amino acid at position 1 to the amino acid at position 144 to the amino acid at position 144 shown in SEQ ID NO: 3 It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to Leu.
  • the present invention also provides (9) a protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 6, wherein the amino acid at position 1 to the amino acid at position 237 shown in SEQ ID NO: 5 It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to Ser.
  • the present invention also relates to (10) a protein encoded by a DNA hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO: 8; It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to the amino acid Ser.
  • the present invention also relates to (11) a protein encoded by a DNA which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 20; It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to amino acid Leu.
  • the present invention also relates to (12) a protein encoded by a DNA hybridizing with DNA comprising the nucleotide sequence of SEQ ID NO: 22, wherein the amino acid at the 1st position and the amino acid at the 538th position are shown in SEQ ID NO: 21. It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to Ser.
  • a fusion protein comprising a protein and another peptide or polypeptide is provided.
  • the present invention also provides (14) a DNA encoding the protein described in any one of (1) to (13) above.
  • the present invention also provides (15) a vector into which the DNA of (14) has been inserted.
  • the present invention also provides (16) a transformant capable of expressing the DNA of (14) above.
  • the present invention also provides (17) a method for producing the protein according to any one of the above (1) to (13), comprising a step of culturing the transformant according to the above (16).
  • the present invention also provides (18) a method for screening a compound that binds to the protein according to any of (1) to (13) above,
  • the present invention also provides (19) an antibody that specifically binds to the protein according to any one of (1) to (12).
  • the present invention also provides (20) contacting the antibody according to (19) with a sample expected to contain the protein according to any one of (1) to (13), A method for detecting or measuring the production of an immunocomplex comprising the protein and a protein.
  • the present invention also provides (21) a DNA which specifically hybridizes with a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 20, and 22 to 27, and has at least 15 nucleotides. DNA having a chain length of
  • the present invention relates to a novel hemopoietin receptor "NR8".
  • NR8 hemopoietin receptor
  • 5 The results of analysis by RACE and 3'-RACE, analysis of NR8 genome sequence, and analysis by plaque screening predicted the presence of NR8, NR8, and NR8a.
  • the structures of these NR8 genes are shown in FIG.
  • NR83 is an alternative splice product lacking the fifth exon, and CDS terminates at the stop codon on the sixth exon that is directly linked to the fourth exon and causes a frame shift. It is possible to encode two different proteins, a soluble protein that binds to a protein and a membrane-bound protein that lacks a signal sequence starting from ATG on the fourth exon.
  • the soluble protein has the same amino acid sequence up to the fourth exon of NR8 and may function as a soluble receptor.
  • NR8a also encodes a protein containing an insertion of 177 amino acids from the NR8 intron 9 near the C-terminus of NR8 as a result of alternative splicing.
  • Both NR8 and NR8 encode a transmembrane hemopoietin receptor.
  • a motif similar to Box1 exists in the vicinity of the cell membrane among sequences conserved among other hemopoietin receptors that are considered to be involved in signal transduction. Since similar sequences exist with low conservation in Box2, NR8 is considered to belong to the type of receptor that transmits signals as homodimers.
  • the amino acid sequence of the protein designated as NR8 contained in the protein of the present invention is represented by SEQ ID NO: 1 (NR8), SEQ ID NO: 3 (soluble NR85), SEQ ID NO: 5 (membrane-bound NR S / 3) and SEQ ID NO: ⁇ (NR8a), and the nucleotide sequences of the cDNA encoding the protein are shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.
  • SEQ ID NO: 2 SEQ ID NO: 4
  • SEQ ID NO: 6 SEQ ID NO: 8
  • NR8 protein described above has potential medical applications. Expression of NR8 in fetal liver, spleen, thymus and certain leukemia cell lines suggests that it may be a receptor for unknown hematopoietic factors. Therefore, NR8 protein is considered to provide a useful material for obtaining this unknown hematopoietic factor.
  • NR8 expression may be specifically expressed in a limited cell population in these hematopoietic tissues, and an anti-NR8 antibody is useful as a means for separating this cell population.
  • the cell population thus separated can be applied to cell transplantation therapy.
  • anti-NR8 antibodies are also expected to be applied to the diagnosis or treatment of leukemia and other diseases.
  • soluble proteins containing the extracellular domain of NR8 protein, or NR8 splice variant NR8 ? are expected to be used as decoy-type receptors as inhibitors of NR8 ligands, including leukemia involving NR8. It can be expected to be applied to the treatment of illness.
  • mouse NR8 cDNA corresponding to the above-mentioned human-derived NR8 cDNA by using the technique of cross-species cross-PCR cloning.
  • the amino acid sequence of the protein designated as mouse NR8 contained in the protein of the present invention is shown in SEQ ID NO: 19 (soluble mouse NR8?) And SEQ ID NO: 21 (mouse NR8a), and encodes the protein.
  • the nucleotide sequences of the cDNA are shown in SEQ ID NO: 20 and SEQ ID NO: 22, respectively.
  • mouse NR8 gene encoding a 538 amino acid transmembrane receptor protein was identified due to differences in the transcripts derived from the splice variant as well as human NR8.
  • mouse NR85 encoding a secreted soluble receptor-like protein consisting of 144 amino acids was confirmed.
  • amino acid sequence encoded by the receptor gene was compared between human and mouse, a high homology of 98.9% was observed in NR8a, and a homology of 97.2 was also observed in NR8a.
  • the expression level of mouse NR8 gene varied by Northern analysis and RT-PCR analysis, expression was observed in all analyzed organs.
  • NR8 of origin its gene expression showed a wide distribution. This suggests that the molecular function of NR8 in mice may be involved in a wide variety of biological physiological regulatory mechanisms.
  • the present invention also encompasses proteins functionally equivalent to the above-mentioned human and mouse NR8 proteins.
  • “functionally equivalent” means that the protein has the same biological activity as the NR8 protein.
  • the biological activity is, for example, hematopoietic factor receptor protein activity.
  • a method for obtaining such a protein a method of introducing a mutation into the amino acid sequence of the protein has been used.
  • a desired mutation can be introduced into an amino acid sequence in a protein by site-directed mutagenesis using a synthetic oligonucleotide primer (Kramer, W. and Fritz, HJ Methods in Enzymol. (1987). ) 154, 350-367).
  • mutations can be introduced into the amino acid sequence in a protein using a site-directed mutagenesis system (manufactured by GIBCO-BRL) by PCR.
  • a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 19, or SEQ ID NO: 21 It is possible to obtain a protein functionally equivalent to the NR8 protein, modified by deletion or addition of one or more amino acids and / or substitution with another amino acid so as not to affect the biological activity. it can.
  • Specific examples of the protein functionally equivalent to the NR8 protein of the present invention include SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 19, and SEQ ID NO: 21
  • SEQ ID NO: 1 One or more, preferably two or more and 30 or less, more preferably two or more and 10 or less amino acids in the amino acid sequence shown in any of SEQ ID NO: 21 are deleted.
  • One or more amino acids in the amino acid sequence, preferably two or more and 30 or less, more preferably two or more and 10 or less amino acids are substituted with other amino acids.
  • a fusion protein may be mentioned as a protein obtained by adding one or more amino acid residues to the NR8 protein of the present invention.
  • the fusion protein is a fusion of the NR8 protein of the present invention and another peptide or protein, and is included in the present invention.
  • the fusion protein is prepared by ligating the DNA encoding the NR8 protein of the present invention and the DNA encoding the other peptide or protein so that their frames match, and introducing this into an expression vector.
  • the expression may be carried out in a host, and a known method can be used.
  • Other peptides or proteins to be fused with the protein of the present invention are not particularly limited.
  • peptides include FLAG (Hopp, TP et al., BioTechnology (1 988) 6, 1204-1210), 6 x His consisting of 6 His (histidine) residues, lO x His, influenza agglutinin (HA) , Human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, single-tubulin fragment, B-tag A known peptide such as a fragment of Protein C is used.
  • proteins for example, GST (glucathione transferase), HA (Influenza agglutinin), immunoglobulin constant region, 3-galactosidase, MBP (maltose binding protein) and the like.
  • GST glucathione transferase
  • HA Influenza agglutinin
  • immunoglobulin constant region immunoglobulin constant region
  • 3-galactosidase MBP (maltose binding protein) and the like.
  • MBP maltose binding protein
  • the protein of the present invention also comprises a DNA consisting of the nucleotide sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20, and SEQ ID NOS: 22 to 27. And a protein that is encoded by DNA that hybridizes under stringent conditions and that is functionally equivalent to the NR8 protein.
  • Stringent conditions include those that can be appropriately selected by those skilled in the art, for example, low stringency conditions.
  • the conditions of low stringency include, for example, 42 ° C., 2 ⁇ SSC, 0.1% SDS, and preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS. More preferably, high stringency conditions can be mentioned.
  • High stringency conditions include, for example, 65 ° C, 2XSSC and 0.1% SDS. Under these conditions, DNA with higher homology can be obtained as the temperature is increased.
  • SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 19 and SEQ ID NO: 21 are functionally equivalent to the above-mentioned NR8 protein.
  • proteins having homology to a protein having any of the amino acid sequences shown in any of them are defined as at least 70%, preferably at least 80%, of the amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. More preferably, it means a protein having at least 90%, more preferably at least 95% or more homology on the amino acid sequence.
  • the algorithm described in the literature Wang, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730 may be used.
  • the protein of the present invention differs in the amino acid sequence, molecular weight, isoelectric point, presence / absence and form of a sugar chain depending on the cell, host, or purification method that produces the protein as described below. However, as long as the obtained protein has hematopoietic factor receptor protein activity, it is included in the present invention.
  • the protein of the present invention when expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the N-terminal of the amino acid sequence of the original protein.
  • a prokaryotic cell for example, Escherichia coli
  • a methionine residue is added to the N-terminal of the amino acid sequence of the original protein.
  • the N-terminal signal sequence is removed.
  • the proteins of the present invention also include such proteins.
  • the signal sequence was determined to be the amino acid sequence of SEQ ID NO: 1 It was estimated that Met from 1st place to Gly in 19th place Accordingly, the present invention includes a protein consisting of Cys at position 20 to Ser at position 361 in the amino acid sequence of SEQ ID NO: 1.
  • the obtained DNA is incorporated into an expression vector so that it can be expressed under the control of an expression control region, for example, an enhancer or promoter.
  • an expression control region for example, an enhancer or promoter.
  • a host cell is transformed with this expression vector to express the protein.
  • promoter / enhancer When using mammalian cells, useful promoters / enhancers commonly used, DNA encoding the protein of the present invention, DNA having a polyA signal functionally linked to its 3 ′ downstream, or DNA Construct a vector containing
  • the promoter / enhancer can be a human cytomegalovirus immediate early promoter / enhancer.
  • promoters that can be used for protein expression include retroviruses, polioviruses, adenoviruses, Simian virus 40 (SV40), and other virus promoters.
  • the promoter (HEFla) derived from mammalian cells may be used.
  • the method of Mulligan et al. (Nature (1979) 277, 108) when using the SV 40 Promoter / Enhancer, and the method of Mizushima et al. (Nucleic (1990) 18, 5322).
  • expression can be performed by operably linking a useful promoter commonly used, a signal sequence for polypeptide secretion, and a gene to be expressed.
  • the promoters include the lacZ promoter and the araB promoter.
  • the lacZ promoter the method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), and when the araB promoter is used, the method of Better et al. (Science (1988) 240, 1041-1043).
  • a pelB signal sequence (Lei, SP. Et al J. Bacteriol. (1987) 169, 4379) may be used when produced by E. coli periplasm.
  • the expression vector can be used as a selection marker to amplify the gene copy number in the host cell system, such as the aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, and Escherichia coli xanthinguanine phosphoribosyltrans. It can contain the ferragase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene, and the like.
  • APH aminoglycoside transferase
  • TK thymidine kinase
  • Escherichia coli xanthinguanine phosphoribosyltrans can contain the ferragase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene, and the like.
  • the expression vector for producing the protein of the present invention may be any expression vector as long as it is an expression vector suitably used in the present invention.
  • the expression vector of the present invention include expression vectors derived from mammals, for example, pEF, pCDM8, expression vectors derived from insect cells, for example, pBacPAK8, expression vectors derived from plants, such as ⁇ 1, pMH2, and expression derived from animal viruses.
  • Vectors such as pHSV, pMV, pAdexLcw, retrovirus-derived expression vectors, such as pZIpneo, yeast-derived expression vectors, such as lb
  • Examples include pNVll, an expression vector derived from SP-QO Bacillus subtilis, such as pPL608, pKTH50, and an expression vector derived from Escherichia coli, such as pQE, pGEAPP, pGEMEAPP, and pMALp2.
  • the vectors of the present invention include those used not only for producing the protein of the present invention in vivo and in vitro, but also for gene therapy of mammals, for example, humans.
  • any production system can be used for protein production.
  • Production systems for protein production include in vitro and in vivo production systems.
  • In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.
  • Animal cells include mammalian cells, such as CHO (J. Exp. Med. (1 995) 108, 945), COS ⁇ myeloma, BHK (baby hamster kidney), HeLa ⁇ Ve ro, amphibian cells, such as African Megafrog Oocytes (Val le, et al., Nature (1981) 291, 358-340) or insect cells such as sf9, sf21, and Tn5 are known.
  • CHO cells in particular, DHFR-deficient CH0 cells such as dhfr-CH0 (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO Kl (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) can be suitably used.
  • yeast for example, the genus Saccharomyces, for example, Saccharomyces cerevisiae, filamentous, lineage; c (Aspergillus), for example, Aspergillus niger (Aspergil lus niger) is known.
  • E. coli Escherichia coli
  • Bacillus subtilis Bacillus subtilis
  • the protein is obtained by transforming these cells with the desired DNA and culturing the transformed cells in vitro.
  • Culture is performed according to a known method.
  • DMEM fetal calf serum
  • FCS fetal calf serum
  • the pH during culturing is preferably about 6-8.
  • Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary.
  • examples of in vivo production systems include production systems using animals and production systems using plants.
  • the desired DNA is introduced into these animals or plants, and proteins are produced and recovered in the animals or plants.
  • the “host” in the present invention includes these animals and plants.
  • mice When using animals, there are production systems using mammals and insects. As mammals, goats, bushes, sheep, mice, and mice can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.
  • a desired DNA is inserted into a gene encoding a protein that is uniquely produced in milk, such as goat casein, to prepare a fusion gene.
  • the DNA fragment containing the fusion gene with the inserted DNA is injected into a goat embryo, and the embryo is introduced into a female goat.
  • the protein is obtained from milk produced by the transgenic goat born from the goat that has received the embryo or its progeny. Hormones may be used in the transgenic goat as appropriate to increase the amount of milk containing protein produced from the transgenic goat. (Ebert, K.M. et al., Bio / Technology (1994) 12, 699-702).
  • silkworms can be used as insects, for example.
  • the baculovirus into which the target DNA is inserted is infected to the silkworm, and a desired protein is obtained from the body fluid of the silkworm (Susumu, M. et al., Nature (1985)). 1 ⁇
  • tobacco when using a plant, for example, tobacco can be used. If tobacco is used, the desired DNA is inserted into a plant expression vector, for example, ⁇ 530, and the vector is introduced into a bacterium such as Agrobacterium m ⁇ efaciens. The bacteria are infected with tobacco, for example, Nicotiana tabacum, to obtain the desired polypeptide from tobacco leaves (Julian, K.-C. Ma et al., Eur. J. Immunol. 1 994) 24, 131-138).
  • tobacco for example, Nicotiana tabacum
  • the protein of the present invention obtained as described above can be isolated from a host inside or outside a cell, and purified as a substantially pure and homogeneous protein.
  • the separation and purification of the protein may be performed by the same separation and purification methods used in ordinary protein purification, and is not limited in any way. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc.
  • the proteins can be separated and purified by appropriately selecting and combining.
  • chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course). Manu al. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HP LC and FPLC. The present invention also includes highly purified proteins using these purification methods.
  • the protein can be arbitrarily modified or partially removed by applying an appropriate protein modifying enzyme before or after purification of the protein. Trypsin, chymotrypsin, lysyl endop Tidase, protein kinase, glucosidase, protein kinase, and glucosidase are used.
  • the present invention also provides an activity of a protein comprising an amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 19, and SEQ ID NO: 21 Including partial peptides consisting of a center.
  • the partial peptides of the protein of the present invention include, for example, partial peptides containing one or more of a hydrophobic region and a hydrophilic region estimated from a hydrophobic plot analysis in a protein molecule. These partial peptides may include part or all of one hydrophobic region, or may include part or all of one hydrophilic region.
  • a soluble protein of the protein of the present invention and a protein comprising an extracellular region are also included in the present invention.
  • the partial peptide of the protein of the present invention can be produced by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase.
  • a genetic engineering technique for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used.
  • the present invention also relates to DNA encoding the protein of the present invention.
  • the cDNA encoding the protein of the present invention can be obtained, for example, by screening a human cDNA library using the probe described in the present specification.
  • cDNA By screening the cDNA library using the obtained cDNA or the cDNA fragment as a probe, cDNA can be obtained from different cells, tissues, organs or species.
  • the cDNA library may be prepared, for example, by the method described in Sambrook, J. et al., Molecular Clonings Cold Spring Harbor Laboratory Press (1989), or a commercially available DNA library may be used.
  • the translation region encoded by the cDNA can be determined, and the amino acid sequence of the protein of the present invention can be obtained. Further, by screening a dienomic DNA library using the obtained cDNA as a probe, dienomic DNA can be isolated. Specifically, the following may be performed. First, mRNA is isolated from cells, tissues, and organs that express the protein of the present invention. The mRNA can be isolated by a known method, for example, guanidine ultracentrifugation (Chirgwin, JM et al. Biochemistry (1979) 18,
  • the total RNA was prepared by the AGPC method (Chomczynski, P. and Sacchi, N Anal. Biochem. (1987) 162 156-159), etc., and the mRNA was purified using the mRNA Purification Kit (Pharmacia). Purify mRNA from total RNA. Also, QuickPrep mMA
  • MRNA can also be directly prepared by using Purification Kit (Pharmacia).
  • CDNA is synthesized from the obtained mRNA using reverse transcriptase.
  • cDNA can also be synthesized using AMV Reverse Transcriptase First-Strand DNA Synthesis Kit (Seikagaku).
  • AMV Reverse Transcriptase First-Strand DNA Synthesis Kit (Seikagaku).
  • the 5′-RACE method (Frohman MA) using the 5 — Ampli FINDER RACE Kit (manufactured by Clontech) and the polymerase chain reaction (PCR) was used. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17 2919-2932) Synthesis and amplification can be performed.
  • a target DNA fragment is prepared from the obtained PCR product, and ligated with the vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector.
  • the nucleotide sequence of the target DNA may be confirmed by a known method, for example, the dideoxynucleotide chain-one-minute method.
  • the DNA of the present invention can be designed to have a higher expression efficiency in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucelic Acids Research (1981)). 9 D43-p74).
  • the DNA of the present invention is ⁇ can be modified by a known method. Examples include digestion with a restriction enzyme, insertion of a synthetic oligonucleotide or an appropriate DNA fragment, addition of a linker, insertion of an initiation codon (ATG) and / or a termination codon (ATT, TGA or TAG), and the like. .
  • the DNA of the present invention is a DNA consisting of the base A at position 441 to the base C at position 1523 in the base sequence of SEQ ID NO: 2, the base sequence of SEQ ID NO: 4 DNA consisting of base A, DNA consisting of base A at position 659 in the base sequence of SEQ ID NO: 6 to base C of position 1368 and base C of base 41 of position 41 to base 2054 in base sequence SEQ ID NO: 8 DNA consisting of base A at position 439 in the base sequence of SEQ ID NO: 20 to base A at position 870 in the base sequence of SEQ ID NO: 20, and base C of base A at position 439 to base 2052 in the base sequence of SEQ ID NO: 22
  • the DNA of the present invention also includes a nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20 and SEQ ID NO: 22 to 27. DNA that hybridizes with other DNAs under stringent conditions and that encodes
  • Stringent conditions can be appropriately selected by those skilled in the art, and examples include low stringent conditions.
  • the conditions of low stringency include, for example, 42 ° C., 2 ⁇ SSC, 0.1% SDS, and preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS. More preferably, high stringency conditions are used.
  • Highly stringent conditions include, for example, 65 ° 2XSSC and 0.1% SDS. Under these conditions, DNA with higher homology can be obtained as the temperature is increased.
  • the hybridizing DNA is preferably a naturally occurring DNA, such as cDNA or chromosomal DNA.
  • the above-mentioned naturally-derived DNA may be, for example, a cDNA dienomic DNA derived from a tissue in which mRNA that hybridizes with the cDNA encoding the protein of the present invention in Examples is detected.
  • the DNA encoding the protein of the present invention may be a synthetic DNA in addition to the cDNA dienomic DNA.
  • the protein of the present invention is useful for screening for a compound that binds to the protein. That is, the protein of the present invention is brought into contact with a test sample expected to contain a compound binding to the protein, and a compound having an activity of binding to the protein of the present invention is selected. Used in methods to screen for compounds that bind to proteins.
  • the protein of the present invention used in the screening of the present invention may be any of a recombinant type, a natural type and a partial peptide.
  • the compound having an activity of binding to the protein of the present invention may be a protein having a binding activity, or may be a chemically synthesized compound having a binding activity.
  • test sample used in the screening method of the present invention includes, for example, peptides, purified or partially purified proteins, non-peptide compounds, synthetic compounds, fermentation products of microorganisms, marine organism extracts, plant extracts, and cell extracts. Liquid and animal tissue extract. These test samples may be novel compounds or known compounds.
  • a cDNA is isolated from a cell, tissue, or organ that is expected to express a protein that binds to the protein of the present invention, and introduced into a phage vector, for example, gtll, ZAPII, etc., to produce a cDNA library.
  • a protein bound to the protein of the present invention which is expressed on a plate from which a medium has been drawn, and the protein of the present invention, which has been expressed and fixed on a plate, is allowed to react with the labeled and purified protein of the present invention, and the above-mentioned filter, and binds to the protein of the present invention. May be detected by labeling the plaques expressing.
  • the method for labeling the protein of the present invention include a method using the binding property of biotin and avidin, and an antibody that specifically binds to the protein of the present invention or a peptide or polypeptide fused to the protein of the present invention.
  • Screening for a ligand that specifically binds to the protein of the present invention comprises linking the extracellular domain of the protein of the present invention with the intracellular domain of the hemopoietin receptor protein having a known signal transduction ability, including the transmembrane domain.
  • a suitable cell line preferably a cell line that can survive and grow only in the presence of a suitable growth factor (growth factor-dependent cell line)
  • growth factor-dependent cell line This can be carried out by culturing the cell line with the addition of a material expected to contain various growth factors, cytodynamic factors, hematopoietic factors and the like.
  • This method utilizes the fact that the growth factor-dependent cell line can survive and proliferate only when the test material contains a ligand that specifically binds to the extracellular domain of the protein of the present invention. are doing.
  • the Mopoechin receptor to known for, for example, Toronbopoechin receptor, erythropoietin receptor, G-CSF receptor, but g P 130, and the like, partners of the chimeric receptor used in the screening system of the present invention, these known
  • the present invention is not limited to the hemopoietin receptor, and any cytoplasmic domain having a structure necessary for signal transduction activity may be used.
  • a growth factor-dependent cell line for example, an IL3-dependent cell line such as BaF3 or FDC-P1 can be used.
  • a rare but soluble protein may be a cell membrane-bound protein.
  • the protein containing only the extracellular domain of the protein of the present invention or the protein containing Screening can be performed by labeling a fusion protein obtained by adding a partial sequence of another soluble protein to the extracellular domain, and then measuring the binding to a cell expected to express the ligand.
  • the protein containing only the extracellular domain of the protein of the present invention include a soluble receptor protein artificially prepared by inserting a stop codon at the N-terminal side of the transmembrane domain, or a soluble NR83 protein Is available.
  • a fusion protein in which a partial sequence of another soluble protein is added to the extracellular domain of the protein of the present invention for example, an Fc site of immunoglobulin, a FLAG peptide or the like is added to the C-terminal of the extracellular domain.
  • the prepared protein can be used.
  • These soluble labeled proteins can also be used for detection in the above-mentioned West Western method.
  • Screening for proteins that bind to the protein of the present invention can also be performed using a two-hybrid system (Fields, S., and Sternglanz, R., Trends. Genet. (1994) 10, 286-292). .
  • an expression vector containing a DNA encoding a fusion protein of the protein of the present invention and one of the subunits of a transcription factor comprising a heterodimer, and a desired cDNA as a test sample and a heterodimer is introduced into cells and expressed.
  • the reporter gene previously constructed in the cell is expressed. Therefore, a protein that binds to the protein of the present invention can be selected by detecting or measuring the expression level of a reporter gene.
  • a DNA encoding the protein of the present invention and a gene encoding the DNA binding domain of LexA are ligated in such a manner that the frames match, and an expression vector is prepared.
  • the expression vector is ligated by linking the desired cDNA to the gene encoding the GAL4 transcription activation domain. -Is prepared.
  • Cells transformed with the HIS3 gene whose transcription is regulated by a promoter that has a LexA-binding motif, are transformed using the two-hybrid system expression plasmid described above, and then transformed into a histidine-free synthetic medium. In short, cell growth is observed only when protein interaction is observed. Thus, it is possible to examine the increase in the expression level of the repo overnight gene depending on the degree of growth of the transformants.
  • luciferase gene for example, luciferase gene, PAI-1 (Plasminogen activator inhibitor typel) gene, ADE2 gene, LacZ gene, CDC25H gene and the like can be used.
  • the two-hybrid system may be constructed by a commonly used method, or may use a commercially available kit.
  • Commercially available two-hybrid system kits include ATCHMARKER Two-Hybrid System, Mammalian MATCHMARKER Two-Hybrid As say Kit (all manufactured by CLONTECH), HybriZAP Two-Hybrid Vector System (manufactured by Stra tagene), CytoTrap two-hybrid system (manufactured by Stratagene). Screening for a protein that binds to the protein of the present invention can also be carried out using affinity chromatography.
  • the protein of the present invention is immobilized on a carrier of an affinity column, and a test sample which is expected to express a protein that binds to the protein of the present invention is applied here.
  • Test samples in this case include cell culture supernatants, cell extracts, cell lysates, and the like. After applying the test sample, the column is washed to obtain the protein bound to the protein of the present invention.
  • the compound isolated by the screening method of the present invention is a candidate for a drug for promoting or inhibiting the activity of the protein of the present invention.
  • Compounds obtained by the screening method of the present invention can be used for humans and other mammals, such as mice, rats, guinea pigs, egrets, chicks, cats, dogs, sheep, bush, sea lions, monkeys, and baboons. When used as a chimpanzee medicament, it can be carried out according to commonly used means.
  • tablets, capsules, elixirs, and microcapsules which are sugar-coated as required, orally, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids
  • a unit dose required for pharmaceutical practice generally recognized as a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, and binder together with a compound having binding activity to the protein of the present invention. It can be produced by mixing in the form. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
  • Excipients that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Swelling agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used.
  • the unit dosage form is a capsule, the above materials may further contain a liquid carrier such as oil and fat.
  • Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
  • Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents, For example, alcohols, specifically ethanol, polyalcohols, such as propylene glycol, polyethylene glyco Or a nonionic surfactant such as polysorbate 80 (TM) or HCO-50.
  • saline isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents, For example, alcohols, specifically ethanol, polyalcohols, such as propylene glycol, polyethylene glyco Or a nonionic surfactant such as polysorbate 80 (TM) or HCO-50.
  • TM polysorbate 80
  • Oily liquids include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. It may also be combined with a buffer, for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • a buffer for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant.
  • the prepared injection solution is usually filled into an appropriate ampoule.
  • the dose of the compound having the binding activity to the protein of the present invention varies depending on the symptom. However, in the case of oral administration, generally, for an adult (assuming a body weight of 60 kg), the dose is from about 0.1 per day. 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
  • the single dose varies depending on the subject of administration, target organ, symptoms, and administration method.
  • parenteral injections usually for adults (with a body weight of 60 kg) per day, It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection.
  • the dose can be administered in terms of the weight per 60 kg.
  • the antibody of the present invention can be obtained as a monoclonal antibody or a polyclonal antibody using known means.
  • An antibody that specifically binds to the protein of the present invention can be obtained by immunizing the protein using the protein as a sensitizing antigen in accordance with a usual immunization method and obtaining the obtained immune cells by a conventional cell fusion method. It can be produced by fusing with a parent cell and screening antibody-producing cells by a usual screening method.
  • a monoclonal or polyclonal antibody that specifically binds to the protein of the present invention may be prepared as follows.
  • the protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to an animal species from which the protein is derived, but may be derived from a mammal such as a human, a mouse or a rat.
  • human-derived proteins are particularly preferred.
  • a human-derived protein can be obtained using the gene sequence or amino acid sequence disclosed herein.
  • the protein used as the sensitizing antigen a protein having the biological activity of any of the proteins described in the present specification can be used.
  • a partial peptide of a protein can also be used. Examples of the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein.
  • antibody refers to an antibody that specifically reacts with the full length or fragment of a protein.
  • the desired protein can be transferred inside or outside the host cell or from the host.
  • a fragment thereof may be obtained by a known method, and this protein may be used as a sensitizing antigen.
  • a cell expressing the protein, a lysate thereof, or a chemically synthesized protein of the present invention may be used as the sensitizing antigen.
  • the mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. ⁇ Egrets and primates are used.
  • mice for example, mice, rats, hamsters and the like are used.
  • a heronoid animal for example, a heron is used.
  • monkeys are used as primates.
  • monkeys of the lower nose for example, cynomolgus monkeys, macaques, baboons, and chimpanzees are used.
  • Immunization of an animal with a sensitizing antigen is performed according to a known method.
  • a sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal.
  • the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and then mixed with an ordinary adjuvant, for example, Freund's complete adjuvant, if desired, and then emulsified.
  • an ordinary adjuvant for example, Freund's complete adjuvant, if desired, and then emulsified.
  • a suitable carrier can be used at the time of immunization with the sensitizing antigen. Immunization is performed in this manner, and an increase in the level of the desired antibody in the serum is confirmed by a conventional method.
  • the blood of a mammal sensitized with the antigen is taken out.
  • the serum is separated from the blood by a known method.
  • a serum containing the polyclonal antibody may be used as the polyclonal antibody, and if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum.
  • the immune cells may be removed from the mammal and subjected to cell fusion.
  • spleen cells are particularly preferable as the immune cells used for cell fusion.
  • the other parent cell to be fused with the immune cell is preferably a mammalian myeloma cell, and more preferably a myeloma cell that has acquired the properties for selecting fused cells by a drug.
  • the cell fusion of the immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46) and the like. It can be performed according to.
  • the hybridoma obtained by cell fusion is selected by culturing in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine).
  • a HAT culture medium a culture medium containing hypoxanthine, aminopterin and thymidine.
  • the cultivation in the HAT culture solution is continued for a period of time sufficient to kill cells (non-fused cells) other than the desired hybridoma, usually several days to several weeks.
  • a conventional limiting dilution method is performed, and screening and cloning of hybridomas producing the desired antibody are performed.
  • human lymphocytes for example, human lymphocytes infected with the EB virus, Sensitize with protein-expressing cells or a lysate thereof, and fuse the sensitized lymphocytes with human-derived myeloma cells having permanent division ability, such as U266, to produce a desired human antibody having protein binding activity Hypri-doma can also be obtained (JP-A-63-17688).
  • a transgenic animal having a human antibody gene repertoire is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain antibody-producing cells, and a hybridoma obtained by fusing the antibody-producing cells with myeloma cells is obtained. May be used to obtain a human antibody against the protein (see International Patent Application Publication Nos. W092-03918, W093-2227, W094-02602, W094-25585, W096-33735, and W096-34096).
  • cells in which immune cells such as sensitized lymphocytes that produce antibodies are immortalized with oncogenes may be used.o
  • the monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a gene recombination technique (for example, Borrebaeck, CAK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBOD IES, Published in the United Kingdom by MACMILLAN PUBL ISHERS LTD, 1990).
  • the recombinant antibody is produced by cloning DNA encoding the antibody from an immune cell such as a hybridoma or a sensitized lymphocyte producing the antibody, incorporating the DNA into an appropriate vector, and introducing the vector into a host.
  • the present invention includes this recombinant antibody.
  • the antibody of the present invention may be an antibody fragment or an antibody modification thereof as long as it binds to the protein of the present invention.
  • an antibody fragment Fab, F (ab ') 2, Fv, or a single chain Fv (scFv) in which an Fv of an H chain and an L chain are linked by an appropriate linker
  • the antibody is treated with an enzyme, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragments is constructed and, after introducing this into an expression vector, an appropriate host Expressed in cells (eg, Co, M. S.
  • modified antibody an antibody bound to various molecules such as polyethylene glycol (PEG) can also be used.
  • PEG polyethylene glycol
  • the “antibody” of the present invention also includes these modified antibodies.
  • Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods have already been established in this field.
  • the antibody of the present invention can be prepared by using a chimeric antibody comprising a non-human antibody-derived variable region and a human antibody-derived constant region or a non-human antibody-derived CDR (complementarity determining region) and a human antibody-derived antibody using known techniques.
  • a chimeric antibody comprising a non-human antibody-derived variable region and a human antibody-derived constant region or a non-human antibody-derived CDR (complementarity determining region) and a human antibody-derived antibody using known techniques.
  • the antibody obtained as described above can be purified to homogeneity.
  • the separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins, and is not limited at all.
  • the concentration of the antibody obtained as described above can be measured by measuring absorbance, enzyme-linked immunosorbent assay (ELISA), or the like.
  • ELISA Enzyme immunoassay
  • RIA radioimmunoassay
  • fluorescent antibody method a fluorescent antibody method
  • the protein of the present invention is added to a plate on which the antibody of the present invention is immobilized, and then a sample containing the target antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added.
  • a secondary antibody that recognizes the antibody labeled with an enzyme for example, alkaline phosphatase, and incubate and wash the plate.
  • antigen binding activity can be evaluated by adding an enzyme substrate such as P-ditrophenyl phosphate and measuring the absorbance.
  • an enzyme substrate such as P-ditrophenyl phosphate
  • a protein fragment for example, a C-terminal fragment or an N-terminal fragment thereof may be used.
  • BIAcore Pharmacia
  • the antibody of the present invention is brought into contact with a sample expected to contain the protein of the present invention contained in the sample, and an immune complex of the antibody and the protein is detected or measured.
  • the method of detecting or measuring the protein of the present invention comprising:
  • the protein detection or measurement method of the present invention can specifically detect or measure a protein, it is useful for various experiments and the like using proteins.
  • the present invention relates to a nucleotide sequence represented by any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20 and SEQ ID NO: 22 to 27 DNA that specifically hybridizes with DNA or DNA complementary to the DNA and has a chain length of at least 15 bases. That is, probes, nucleotides or nucleotide derivatives capable of selectively hybridizing DNA encoding the protein of the present invention or DNA complementary to the DNA, such as antisense oligonucleotides and ribozymes, are included.
  • the present invention relates to, for example, a base represented by any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20 and SEQ ID NO: 22 to 27
  • This antisense oligonucleotide preferably has SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20 and any one of SEQ ID NOs: 22 to 27. It is an antisense oligonucleotide to at least 15 or more consecutive nucleotides in the nucleotide sequence shown. More preferably, the antisense oligonucleotide described above, wherein the continuous at least 15 or more nucleotides include a translation initiation codon. ⁇
  • antisense oligonucleotide derivatives and modifications thereof can be used.
  • modifications include lower alkylphosphonate modifications such as methylphosphonate type or ethylphosphonate type, phosphorothioate modifications and phosphoramidite modifications.
  • antisense oligonucleotide includes not only those in which all the nucleotides corresponding to the nucleotides constituting the predetermined region of DNA or mRNA are complementary, but also those in which DNA or mRNA and the oligonucleotide are sequence numbers: A mismatch of one or more nucleotides may exist as long as it can selectively and stably hybridize to the base sequence shown in 1.
  • Selectively and stably hybridizing is defined as cross-hybridization with DNA encoding other proteins under normal hybridization conditions, preferably under stringent hybridization conditions. Does not occur significantly.
  • DNAs should have at least 15 contiguous nucleotide sequence regions with at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95% or more nucleotide sequence homology. Show what you have.
  • the algorithm for determining homology may use the algorithm described in this specification.
  • Such a DNA is useful as a probe for detecting or isolating a DNA encoding the protein of the present invention, or as a primer for amplifying, as described in Examples below.
  • the antisense oligonucleotide derivative of the present invention acts on cells producing the protein of the present invention to inhibit transcription or translation of the protein by binding to DNA or mRNA encoding the protein, By suppressing the expression of the protein of the present invention, for example, by promoting the degradation of the protein, the effect of suppressing the action of the protein of the present invention is obtained.
  • the antisense oligonucleotide derivative of the present invention can be used as an external preparation such as a liniment or a poultice by mixing with a suitable base material which is inactive against the derivative. If necessary, excipients, isotonic agents, solubilizing agents, stabilizers, preservatives, soothing agents, etc. are added to tablets, splinters, granules, capsules, liposomal capsules, and injections. Agents, solutions, nasal drops, etc., and further can be lyophilized. These can be prepared according to a conventional method.
  • the antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is applied to the patient so that it can reach the affected area as a result of intravenous administration or the like.
  • an antisense-encapsulated material that enhances durability and membrane permeability can be used.
  • ribosome, poly-L-lysine, ribid, cholesterol, lipofectin or derivatives thereof can be mentioned.
  • the dosage of the antisense oligonucleotide derivative of the present invention can be appropriately adjusted according to the condition of the patient, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably 0.1 to 50 mg / kg.
  • the antisense oligonucleotide of the present invention inhibits the expression of the protein of the present invention, and is therefore useful in suppressing the biological activity of the protein of the present invention.
  • Expression inhibition containing the antisense oligonucleotide of the present invention The agent is useful in that it can suppress the biological activity of the protein of the present invention.
  • FIG. 1 is a schematic diagram showing the result of a BlastX search using 180 bases of 40952-40966 including the only probe sequence 40952-40966 in the AC002303 sequence as a query. “#”: The number is indicated by nucleotide number only for NR8. An underline attached to the NR8 sequence indicates a portion corresponding to exon. Other underlined sequences indicate identical amino acids.
  • Figure 2 is a schematic diagram showing the result of B lastX scan of 180 bases in both 5 'and 3' directions, centering on 180 bases of 409 52-40966 including the only probe sequence 40952-40966 in the AC002303 sequence.
  • Figure 3 shows the results of RT-PCT amplification using human fetal liver and skeletal muscle cDNA as type III, amplified by a combination of SN1 / AS1, SN1 / AS2, and SN2 / ASK SN2 / AS2 primers.
  • Figure shows the time.
  • FIG. 4 shows the results of electrophoresis of the results of performing the 5′-RACE method and the 3′-RACE method using human fetal liver cDNA as type II.
  • FIG. 5 shows the nucleotide sequence and amino acid sequence of the cDNA of NR8.
  • the positions of the primers used for RT-PCR are indicated by arrows. In order from the 5 'side, they are SN1 (798-827), SN2 (894-923), AS2 (1055-1026), and AS1 (1127-1098). However, for AS1, the genome-derived AC was used instead of CT for the two bases at the 5 'end.
  • FIG. 6 is a continuation of FIG. 5 showing the nucleotide sequence and amino acid sequence of cDNA of NR8.
  • FIG. 7 shows the nucleotide sequence and amino acid sequence of NR8? CDNA. Two possible open reading frames (0RF) are shown.
  • FIG. 8 is a continuation of FIG. 7 showing the nucleotide sequence and amino acid sequence of the cDNA of FIG.
  • FIG. 9 is a diagram showing the nucleotide sequence and amino acid sequence of cDNA of NR8. The 177 amino acids inserted by alternative splicing are underlined.
  • FIG. 10 is a continuation of FIG. 9 showing the nucleotide sequence and the amino acid sequence of the NR8 cDNA.
  • C Figure 1 1 which underlined 177 amino acids inserted by alternative splicing are continuation of FIG. 1 0 shows the nucleotide sequence and amino acid sequence of the cDNA of NR87.
  • FIG. 12 is a diagram showing the results of analyzing the expression of NR8 in each organ by Northern plotting.
  • FIG. 13 is a diagram schematically showing the structure of the NR8 gene.
  • Other iterations include (CA) n, (CAGA) n, (TGGA) n, (CATA) n, (TA) n, (GA) n, (GGAA) n, (CATG) n, (GAM) n , MSTA, AT-rich, LT1A1, LINE2, FLAM-C, MER63A, MSTB.
  • FIG. 14 shows a schematic structural diagram of an expressible protein constructed in an expression vector.
  • FIG. 15 shows the results of cross-PCR using a human NR8 primer set against a mouse cDNA library.
  • a 100 bp DNA Ladder (NEB # 323-1L) was used as a size marker.
  • FIG. 16 shows the results of comparing the amino acid sequences of human and mouse NR83. The amino acid sequence where the two match was colored. In the sequence, cysteine residues conserved in other hemopoietin receptors are shown in bold.
  • FIG. 17 shows the results of comparing the amino acid sequences of human and mouse NR8a. The amino acid sequence where the two match was colored. In the sequence, cysteine residues conserved in other hemopoietin receptors and WSXWS-Box are shown in bold.
  • FIG. 18 shows the results of analyzing the expression of the NR8 gene in each mouse organ by RT-PCR.
  • 100 bp DNA Ladder (NEB # 323-1L) was used as a size marker, and indicated at both ends of the lane.
  • a 320 bp target gene was detected in all organs.
  • Figure 19 shows the results of analyzing the expression of the NR8 gene in each mouse organ by Northern blotting (left). Only in the testis, a transcript of about 4.2 kb was strongly detected. As a positive control, we also detected mouse whole actin of the same plot (right).
  • oligonucleotide As an oligonucleotide encoding the Trp-Ser-Xaa-Trp-Ser motif, 256 probe sequences consisting of tggag (t / c) nnntggag (t / c) (n is an arbitrary base) are designed. O 99/67290 ⁇ PC first 9 strokes 51
  • TGGAGCATATGGAGT lie (:. 0020 r) 2 28882 tggagcatatggagt 28896 9p22 myosin VIIA,
  • AC002303, AC003112, AL008637 and AC004004 hit multiple known hemopoietin receptors, but AC004004 was a stop codon 3 amino acids downstream of the Trp-Ser-Xaa-Trp-Ser motif. Was excluded from the existence.
  • AL008637 was considered to be GM-CSF receptor 5, a known receptor.
  • AC002303 is a BAC clone CIT987-SKA-670B5 derived from the 16pl2 region of human chromosome 16, which was registered on June 19, 1997 by the TI GR group and has a total length of 1315 30 base pairs (Lamerdin, J. E., et al. ., GenBank Report on AC003112, 1 997) o
  • BlastX search using 180 bases of 40861-41040 including tggagtgaatggagt (40952-40966), which is the only probe sequence in the AC002303 sequence A number of the first hemopoietin receptors showed obvious homology, but no known hemopoietin receptor that perfectly matched the query sequence was registered in the database.
  • a 180-base sequence was sequentially cut in both 5 'and 3' directions centering on the 180-base sequence described above, which was used as a query, and a BlastX scan was performed under the above conditions. Sequences exhibiting homology to the receptor were found in the regions 39181-39360 and 4230-42480, and were considered to be other exons of the same gene (Fig. 2).
  • the Pro-rich motif PAPPF was conserved at 39181-39360, and the Box 1 motif was conserved at 42301-42480.
  • the 3 'exon adjacent to the exon containing the Trp-Ser-Xaa- Trp- Ser motif has a transmembrane domain, but this domain has low homology to other hemopoietin receptors and is detected by BlastX scan. What. The above results suggested that the BAC clone C IT987-SKA-670B5 may have a novel hemopoietin receptor gene.
  • NR8-SN1 5'-CCG GCT CCC CCT TTC AAC GTG ACT GTG ACC-3, (SEQ ID NO: 9)
  • NR8-SN2 5'-GGC AAG CTT CAG TAT GAG CTG CAG TAC AGG-3, (SEQ ID NO: Ten )
  • RT-PCR was performed using the combination of the above primers.
  • the PCR was performed using Advantage cDNA Polymerase Mix (Clontech # 8417-l) and a Perkin Elmer Gene Amp PCR System 2400 thermal cycler under the following experimental conditions. That is, PCR conditions were 4 cycles at 94 ° C, 5 cycles of ⁇ 20 seconds at 94 ° C, 3 minutes at 72 ° C '', and 5 cycles of ⁇ 20 seconds at 94 ° C, 3 minutes at 70 ° C ''. The cycle, "20 seconds at 94 ° C, 3 minutes at 68 ° C", is 28 cycles, 4 minutes at 72 ° C, and ends at 4 ° C.
  • the obtained PCR product was subcloned into pGEM-T Easy vector (Promega # A1360), and the nucleotide sequence was determined.
  • the recombination of the PCR product into the pGEM-T Easy vector was performed at 4 ° C. for 12 hours using T4 DNA Ligase (Promega # A1360).
  • Transgenic products of the PCR product and the pGEM-T Easy vector were obtained by transforming E. coli strain DH5a (Toyobo # DNA-903).
  • Insert Check Ready (Toyobo # PIK-101) was used for selecting the recombinants. Further, the nucleotide sequence was determined using ABI PRISM 377 DNA Sequencer using dRhodamine Terminator Cycle Sequencing Kit (ABI / Perkin Elmer # 4303141). As a result of determining the nucleotide sequence of all the insert fragments for 10 independent clones of the recombinant, all the clones showed a single nucleotide sequence. It was confirmed that the obtained sequence was a partial nucleotide sequence of NR8.
  • the primary PCR conditions were 94 ° C for 4 minutes, 5 cycles of ⁇ 20 seconds at 94 ° C, 4 minutes at 72 ° C '', ⁇ 9 5 cycles of ⁇ 20 seconds at 4 ° C, 4 minutes at 70 ° C '', 28 cycles of ⁇ 20 seconds at 94 ° C, 4 minutes at 68 ° C '', 4 minutes at 72 ° C, and 4 ° C Is over.
  • the conditions for the secondary PCR were as follows: 4 cycles at 94 ° C, 5 cycles of ⁇ 20 seconds at 94 ° C, 3 minutes and 30 seconds at 70 ° C '', ⁇ 20 seconds at 94 ° C, 3 minutes at 68 ° C 30 Seconds ”for 28 cycles, 4 minutes at 72 ° C, and termination at 4 ° C.
  • both of the obtained two types of PCR products were subcloned into the pGEM-T Easy vector, and the nucleotide sequences of all the insert fragments were determined for 16 independent gene recombinants.
  • the nucleotide sequence was determined using the dRhodamine Terminator Cycle Sequencing Kit and analyzed using the ABI PRISM 377 DNA Sequencer as described above. As a result, it was possible to distinguish between 14 clones and 2 clones based on the differences in base pair length and sequence (this will be described later. The difference is that a group of 14 independent clones contains a sequence corresponding to exon 5 in the genomic sequence, and a group of two independent clones does not include this sequence.)
  • 3 ′ RACE-PCR was attempted using the NR8-SN1 primer for primary PCR and the NR8-SN2 primer for secondary PCR.
  • Human Fetal Liver Marathon-Ready cDNA Library was used as type ⁇ , and Advantage cDNA Polymerase Mix was used for PCR experiments.
  • a single-band PCR product was obtained.
  • the obtained PCR product was subcloned into the pGEM-T Easy vector in the same manner as described above, and the nucleotide sequence of all the insert fragments was determined for 12 independent clones of the gene recombinant.
  • the nucleotide sequence was determined by the ABI PRISM 377 DNA Sequencer using the dRhodamine Terminator Cycle Sequencing Kit as described above. As a result, all 12 independent clones showed a single nucleotide sequence.
  • the 0RF of NR8 cDNA is considered to be the start codon based on the presence of an in-frame (39 frame) upstream in-frame stop codon starting from base number 441. Ends with two stop codons starting at 1524.
  • a typical secretory signal sequence a domain considered to be a ligand binding site containing a Cys residue conserved in other hemopoietin receptor members, Pro-ritzimochi, Trp-Ser-Xaa-Trp It has features of hemopoietin receptor such as -Ser motif, transmembrane domain, Boxl motif thought to be involved in signal transduction.
  • NR8 J3 I-crine l + 2 + 3 + 4 + 6 + 7 + 8 + 9a + 10 (two alternatives for soluble and transmembrane (-signal): T reading frame)
  • the full-length NR8 protein prepared based on all the cDNA fragments obtained in Example 3 Using the cDNA as a probe, Northern analysis was performed. Preparation of probe, using the Mega Prime Kit (Amersham, cat # RPN1607), [ shed - 32 P] labeled by dCTP (Amersham, cat # AA0005) .
  • the Northern blot used Human Multiple Tissue Northern (MTN) Blot (Clontech # 7760-1), Human MTN Blot IV (Clontech # 7766-l), and Human Cancer Cell Line MTN Blot (Clontech # 7757-l).
  • Hybridization used Express Hyb Hybridization Solution (Clontech # 8015-2).
  • Hybridization conditions were 68 ° C / 30 minutes of prehybridization followed by 68 ° C / 14 hours of hybridization. After washing under the following conditions, the plate was exposed to an imaging plate (FUJI # BAS-III), and the gene expression of NR8 mRNA was detected by an Image Analyzer (FUJIX, BAS-2000 II).
  • the washing conditions were (1) lx SSC I 0.1% SDS, 5 minutes at room temperature, (2) lx SSC / 0.1% SDS, 30 minutes at 50 ° C, (3) O.lx SSC I 0.13 ⁇ 4 SDS, 50. 30 minutes in C.
  • the obtained PCR product was subcloned into the pGEM-T Easy vector in the same manner as described above, and the nucleotide sequence of the insert fragment was determined.
  • the nucleotide sequence was determined using the ABI PRISM 377 DNA Sequencer using the BigDye Terminator Cycle Sequencing SF Ready Reaction Kit (ABI / Perkin Elmer # 4303150).
  • ABI PRISM 377 DNA Sequencer using the BigDye Terminator Cycle Sequencing SF Ready Reaction Kit (ABI / Perkin Elmer # 4303150).
  • the plasmid containing the NR8 cDNA (SEQ ID NO: 8) was named PGEM-NR8, and E. coli containing the plasmid was identified on October 9, 1998 by the Institute of Industrial Science and Technology. It has been deposited internationally under the Budapest Treaty under the deposit number FERM BP-6545 at the Research Institute for Biotechnology Industry (Tsukuba-Higashi 1-3-1, Ibaraki Prefecture).
  • nucleotide sequences of 3UTR-1, 3UTR-2 and 3UTR-3 as calculated from the translation termination codon of the NR8 cDNA sequence are represented by SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively. It was shown to.
  • NR8 a nucleotide sequence of 3UTR-B1, and 3 UTR-B3 in the case of counting from the translation termination codon of the cDNA sequence, it it SEQ ID NO: 2 6 and SEQ ID NO: 2 7 c Table 1 0 shown in
  • the NR8 gene transcript could encode various different sizes depending on the length of the 3′-terminal untranslated region sequence as well as the difference in alternative splicing. . This may fully explain the presence of transcripts of various sizes, detected by Northern analysis.
  • a screening system was constructed to search for a ligand capable of specifically binding to NR8, ie, a novel hemopoietin.
  • a ligand capable of specifically binding to NR8, ie, a novel hemopoietin was amplified by PCR, and this DNA fragment was A fusion sequence encoding a chimeric receptor was prepared by binding in frame with a DNA fragment encoding the transmembrane region and the intracellular region of the hemopoietin receptor.
  • the human TP0 receptor Human MPL-P
  • Fig. 14 shows a schematic diagram of the structure of the constructed NR8 / TP0-R chimera receptor. Furthermore, the base sequence of the constructed chimeric receptor and the expressible amino acid sequence encoded by it are shown in SEQ ID NOs. : Shown in 13 and 14.
  • the NR8 / TP0-R chimeric receptor expression vector was introduced into the growth factor-dependent cell line Ba / F3 together with the expression vector pSV2bsr (manufactured by Kaken Pharmaceutical Co., Ltd.) containing the blasticidin S resistance gene and forcedly expressed. Thereafter, the transduced cells were selected by culturing in the presence of 8 ⁇ g / ml blasticidin hydrochloride S (manufactured by Kaken Pharmaceutical Co., Ltd.) and IL3. When the resulting chimeric receptor-introduced cells are switched to the absence of IL-3, and a material that is expected to contain the target ligand is added and cultured, and a ligand that specifically binds to NR8 is present. Screening using viability / proliferation is only feasible.
  • NR8 / IgG1-Fc soluble fusion to search for cell membrane-bound ligands or to detect soluble ligands by BIAcore (Pharmacia) or West Western method.
  • a synthetic protein was prepared. 5) The DNA fragment encoding the extracellular region (amino acid sequence; from Met at position 1 to Glu at position 228) of NR8 prepared in section -1 was ligated to the Fc region of human immunoglobulin IgGl. A fusion sequence encoding the soluble fusion protein was prepared by in-frame binding to the encoding DNA fragment.
  • a schematic diagram of the structure of the soluble fusion protein encoded by the constructed NR8 / IgGl-Fc is shown in Fig.
  • SEQ ID NOS: 15 and 1 SEQ ID NOS: 14 and its nucleotide sequence and the expressible amino acid sequence encoded by it are shown in SEQ ID NOS: 15 and 1, respectively. See Figure 6.
  • the fusion gene fragment was inserted into a plasmid vector that can be expressed in mammalian cells, and the constructed expression vector was named pEF-NR8 / IgGl-Fc. After forcibly expressing this pEF-NR8 / IgGl-Fc in mammalian cells and selecting stable transfected cells, the recombinant protein secreted in the culture supernatant was purified using an anti-human IgGl-Fc antibody. It can be purified by immunoprecipitation or affinity column.
  • Recombinant NR8 protein was prepared to search for cell membrane-bound ligand or to detect soluble ligand by BIAcore (Pharmacia) or West Western method.
  • the termination codon was replaced with a nucleotide sequence encoding any amino acid residue by point mutation, and then linked to the nucleotide sequence encoding the FLAG peptide in in-frame.
  • the ligated fragment was inserted into a plasmid vector that can be expressed in mammalian cells, and the constructed expression vector was named PEF-B0S / NR8-FLAG.
  • a schematic diagram of the structure of the inserted fragment NR8-FLAG in the constructed expression vector is shown in FIG.
  • nucleotide sequence of FLAG and the expressible amino acid sequence encoded thereby are shown in SEQ ID NOs: 17 and 18.
  • the recombinant protein secreted into the culture supernatant is immunized with an anti-FLAG peptide antibody. Sedimentation can be performed, or purification can be performed using an affinity column or the like.
  • SEQ ID NOS: 9 and 10 sense side
  • SEQ ID NOS: 11 and 12 upstream direction
  • four types of primer sets can be constructed. That is, using the combination of [NR8-SN1 vs. brain -AS1] ⁇ [NR8-SN1 vs. NR8-AS2], [NR8-SN vs.
  • the mouse brain Amplification of the cross-PCR product was expected by performing the cDNA (Clontech # 7450-1) and mouse testis cMA (Clontech # 7455-l) library type 1 as type I.
  • Advantage cDNA Polymerase Mix (Clontech # 8417-l) was used for the PCR reaction.
  • a Perkin Elmer Gene Amp PCR System 2400 Thermal Cycler under the following cycle conditions, an attempt was made to amplify a partial base sequence capable of encoding a mouse homologous gene of the receptor.
  • the cross-PCR conditions were as follows: 4 minutes at 94 ° C, 5 cycles of ⁇ 20 seconds at 94 ° C, 1 minute at 72 ° C '', 5 cycles of ⁇ 20 seconds at 94 ° C, 1 minute at 70 ° C '' 5 cycles, 28 cycles of “20 seconds at 94 ° C, 1 minute at 68 ° C”, 72 cycles. Terminate at 4 ° C for 4 minutes and at 4 ° C.
  • a mouse brain cDNA-derived product was subcloned into pGEM-T Easy vector (Promega # A1360) to determine the nucleotide sequence.
  • Recombination of the PCR product into pGEM-T Easy vector is performed using T4 DNA Ligase (Promega # A1360). The reaction was carried out at 4 ° C for 12 hours.
  • the PCR product and the recombinant of pGEM-T Easy vector were obtained by transforming E. coli strain DH5 (Toyobo # DNA_903).
  • the recombinants were selected using Insert Check Ready Blue (Toyobo # PIK-201).
  • nucleotide sequence was determined using the ABI PRISM 377 DNA Sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI / Perkin Elmer # 4303154). As a result of determining the nucleotide sequence of all the insert fragments in eight independent clones of the recombinant, a nucleotide sequence derived from the same transcript was obtained, and it was confirmed that both were partial nucleotide sequences of mNR8 . The obtained partial base sequence is shown in SEQ ID NO: 28.
  • an oligonucleotide primer specific to mouse NR8 was designed. As shown below, mNR8-SN3 was synthesized on the sense side (downstream) and mNR8-AS3 was synthesized on the antisense side (upstream). The primer was synthesized using ABI 394 DNA / RNA Synthesizer under the condition of adding a 5'-terminal trityl group. Thereafter, the full-length synthetic product was purified using an OPC column (ABI # 400771). These primers were provided for the 5'-RASE and 3'-RACE methods described below.
  • the NR8-AS2 primer (SEQ ID NO: 12) was used for primary PCR, and the mNR8-AS3 primer (SEQ ID NO: 30) described in the previous section was used for secondary PCR. 5, -RACE PCR was attempted.
  • Mouse Brain arathon-Ready cDNA Library (Clontech # 7450-1) was used as type ⁇ , and Advantage cDNA was used for PCR experiments.
  • the conditions for secondary PCR were 4 minutes at 94 ° C, 5 cycles of ⁇ 20 seconds at 94 ° C, 100 seconds at 70 ° C '', and 5 cycles of ⁇ 20 seconds at 94 ° C and 100 seconds at 68 ° C ''. 25 cycles, 3 minutes at 72 ° C, and termination at 4 ° C.
  • Both of the obtained two PCR products were subcloned into the pGEM-T Easy vector as described above, and the nucleotide sequences were determined.
  • the recombination of the PCH product into the pGEM-T Easy vector was performed at 4 ° C for 12 hours using T4 DNA Ligase.
  • PCR products and recombinants of the pGEM-T Easy vector were obtained by transforming E. coli strain DH5.
  • Insert Check Ready Blue was used in the same manner as described above.
  • the analysis was carried out by ABI PRI SM 377 DNA Sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit.
  • the NR8-SN1 primer (SEQ ID NO: 9) was used for primary PCR, and the mNR8-SN3 primer (SEQ ID NO: 29) was used for secondary PCR.
  • -RACE PCR was attempted.
  • Mouse Brain Marathon-Ready cDNA Library was used as type ⁇ , and Advantage cDNA Polymerase Mix was used for PCR experiments.
  • a Perkin Elmer Gene Amp PCR System 2400 thermal cycler under the same PCR conditions as above, a PCR product showing a single size was obtained. The obtained PCR product was subcloned into the pGEM-T Easy vector according to the description in 7-2), and the nucleotide sequence was determined.
  • the nucleotide sequence of all the insert fragments was determined for four independent recombinants, and it was confirmed that they contained the C-terminal sequence of the full-length mNR8 cDNA clone.
  • the nucleotide sequence determined as a result of the 3′RACE-PCR with the nucleotide sequence of the 5′RACE-PCR product determined in the above 7-4), the final full-length mNR8 ⁇ , and The entire nucleotide sequence of the full-length mNR8 cDNA was determined.
  • the determined nucleotide sequence of the mNR8 cDNA and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 22 and 21, respectively.
  • the determined nucleotide sequence of mNR8 ⁇ cDNA and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 20 and 19.
  • FIG. 16 shows the results of comparing the amino acid sequences of human and mouse NR8 ?. The results of comparing the amino acid sequences of human and mouse NR8a are shown in FIG.
  • Cys at position 25 to Cys residue at position 35 is a typical ligand binding site sequence, and the Cys residues at positions 65 and 109 are common to other hemopoietin receptor members.
  • 1 shows the conserved Cys residue repeat structure.
  • the Pro-rich region is subsequently conserved by Pro residues at positions 120 and 122 and 123, and a typical WSXWS from Trp at position 2 14 to Ser at position 218.
  • -box WS motif
  • Pro residues at positions 271 and 273 in the intracellular region immediately after that are Box-1 consensus sequences (PXP motifs) that are well conserved among other hemopoietin receptor members, and this is signal transduction. It is thought to be deeply involved in As described above, mNR8a fully satisfies the characteristics of one member of the hemopoietin receptor.
  • the exon sequence coding for the pro-rich region is skipped by alternative splicing and directly connected to the next exon site encoding the WS motif.
  • the frame shift shifts the WSXWS-box sequence out of reading frame, encoding up to Leu residue at position 144, and then terminating the translation frame with the next stop codon. This encodes a soluble hemopoietin receptor-like protein that does not have a transmembrane domain.
  • mMA was detected by RT-PCR analysis.
  • primers used for RT-PCR analysis NR8-SN1 primer (SEQ ID NO: 9) was selected as the primer on the sense side (downstream direction), and NR8-AS1 Primer was selected as the primer on the antisense side (upstream direction).
  • the mouse Multiple Tissuue cDNA Panel (Clontec h # K1423-1) was used as type ⁇ .
  • PCR was performed using an Advantage cDNA Polymerase Mix (Clontech # 8 417-1) and a Perkin Elmer Gene Amp PCR System 2400 thermal cycler. The PCR reaction was performed under the following cycle conditions to try to amplify the target gene.
  • the PCR conditions were 4 cycles at 94 ° C, 5 cycles of ⁇ 20 seconds at 94 ° C, 1 minute at 72 ° C '', 5 cycles of ⁇ 20 seconds at 94 ° C, 1 minute at 70 ° C '', 20 cycles at 94 ° C, 1 minute at 68 ° C ”are 24 cycles, 3 minutes at 72 ° C, and end at 4 ° C.
  • RT-PCR results showed that strong expression of the gene was detected in testis and day 17 embryos, and that constitutive mRNA was analyzed in all mouse organs and tissues analyzed. Gene expression was observed.
  • the copy number of type mRNA mRNA was previously normalized between samples. Standardized).
  • the size of the RT-PCR amplification product detected here was 320 bp, which matches the size calculated from the determined base sequence. Therefore, these were considered to be products of mouse NR8-specific PCR amplification reaction.
  • the PCR product amplified in the 17-day embryo was subcloned into pGEM-T Easy vector according to the above 7-2), and the nucleotide sequence was analyzed. As a result, it was confirmed to be a partial nucleotide sequence of mouse NR8, and the possibility of being a product of non-specific PCR amplification was denied.
  • Hyb-ridization Solution (Clontech # 8015-2) is used for hybridization, and after pre-hybridization at 68 ° C / 30 minutes, heat-denatured labeled probe is added, and 68 ° C / 16 hours Hybridization was carried out. After washing under the following conditions, the plate was exposed to an imaging plate (FUJI # BAS-III), and a mouse NR8-specific signal was detected with an image analyzer (FUJIX, BAS-2000II).
  • the washing conditions were (1) lx SSC I 0.13 ⁇ 4 SDS, 5 minutes at room temperature, (2) lx SSC / 0.13 ⁇ 4 SDS, 30 minutes at 50 C, (3) 0.5x SSC / 0.13 ⁇ 4 SDS, 50 ° C In 30 minutes.
  • Example 9 Isolation of NR8 mouse genomic gene by plaque screening The present inventors next attempted plaque hybridization against a mouse genomic DNA library with the aim of analyzing the genomic structure of the mouse NR8 gene.
  • a c library 129SVJ strain Genomic DNA (Stratagene # 946313) constructed in Lambda F IX II was used.
  • a genomic library of about 5.0 ⁇ 10 5 plaques was developed, and plotted on a charged nylon membrane with Hybond N (+) (Amersham # RPN303B), and provided for primary screening.
  • the probe used was the NR8 cDNA fragment of the 5'-RACE product obtained in 7-4).
  • the washing conditions were (1) lx SSC / 0.1% SDS, 5 minutes at room temperature, (2) lx SSC / 0.1% SDS, 30 minutes at 58 ° C, (3) 0.5x SSC / 0.1 % SDS, 30 minutes at 58 ° C.
  • the present invention provides a novel hemopoietin receptor protein "NR8" and a DNA encoding the same. Also provided are a vector into which the DNA has been inserted, a transformant carrying the DNA, and a method for producing a recombinant protein using the transformant. Further, a method for screening a natural ligand or compound that binds to the protein was provided.
  • the NR8 protein of the present invention is considered to be related to hematopoietic action, and is useful for analyzing hematopoietic action. It is also expected to be applied to diagnosis and treatment of hematopoietic diseases.
  • mouse NR8 protein Since the expression of the mouse NR8 gene showed a wide distribution in mouse organs, there is a possibility that the mouse NR8 protein is involved in a wide variety of biological physiological regulatory mechanisms including the above-mentioned hematopoietic action.
  • the mouse NR8 protein it is possible to first isolate a mouse NR8 ligand, and then to isolate a human homologous gene of the NR8 ligand using the conserved structure of the mouse NR8 ligand. Specifically, after determining the nucleotide sequence of mouse NR8 ligand cDNA, oligonucleotide primers are designed on the sequence, and cross-PCR is performed by using them and using a human-derived cDNA library as a ⁇ type.
  • human NR8 ligand cDNA can be obtained by performing cross-hybridization on a human-derived cDNA library using mouse NR8 ligand cDNA as a probe. Further, by using the mouse NR8 gene to generate a mouse lacking the mouse NR8 gene, it is possible to further analyze the biological function of the NR8 receptor protein.

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Abstract

Novel hemopoietin receptor proteins (proteins having the amino acid sequences represented by SEQ ID NOS: 1, 3, 5, 7, 19 and 21); proteins having amino acid sequences derived from the amino acid sequences of the above proteins by modification via deletion, addition or substitution of one or more amino acids; genes encoding these proteins; a process for producing these proteins; and utilization of these proteins and genes.

Description

明細 新規へモポェチン受容体蛋白質 技術分野  Description New hemopoietin receptor protein
本発明は新規へモポェチン受容体蛋白質、 それをコードする遺伝子、 それら の製造方法及び用途に関する。 背景技術  The present invention relates to a novel hemopoietin receptor protein, a gene encoding the same, a method for producing the same, and a use thereof. Background art
種々の細胞の増殖分化、 あるいは分化成熟した細胞の機能の賦活化さらには 細胞死に関与する体液性因子として数多くのサイ トカインの存在が知られてい る。 これらのサイ ト力インにはそれそれ特異的な受容体が存在し、 これらの受 容体は構造上の類似性から幾つかのフアミ リーに分類されている（Hilton D.J. , in "Guidebook to Cytokines and Their Receptors" eaited by Nicola N.A. (A Sambrook & Tooze Publication at Oxford University Press ) , 1994, p8- 16) 。  Many cytokines are known as humoral factors involved in the proliferation and differentiation of various cells or the activation of differentiated and mature cells and the cell death. Each of these site receptors has specific receptors, and these receptors have been classified into several families based on their structural similarities (Hilton DJ, in "Guidebook to Cytokines and Their Receptors "eaited by Nicola NA (A Sambrook & Tooze Publication at Oxford University Press), 1994, p8-16).
一方、 受容体間の類似性と比較するとサイ トカイン同士の一次構造上の相同 性は低く、 同一の受容体フアミ リーに属するサイ トカインメンバ一の間でさえ アミノ酸レベルでの顕著な相同性は認められない。 このことは個々のサイ トカ インの機能の特異性を説明すると同時に、 個々のサイ トカインにより誘導され る細胞の反応の類似性を説明する。  On the other hand, compared with the similarity between receptors, homology in the primary structure between cytokines is low, and remarkable homology at the amino acid level is recognized even among cytokine members belonging to the same receptor family. I can't. This explains the specificity of the function of individual cytokines, as well as the similarity of cellular responses induced by individual cytokines.
上記サイ トカイン受容体フアミ リーの代表的なものとして、 チロシンキナー ゼ受容体、 へモポェチン受容体、 腫瘍壊死因子（TNF )受容体、 トランスフォーミ ング増殖因子/? (TGF ? )受容体の各フアミ リーが挙げられ、それそれのフアミリ 一で異なるシグナル伝達系の関与が報告されている。 これらの受容体ファミリ —のうち、 特にへモポェチン受容体フアミ リーの多くは血液細胞あるいは免疫 担当細胞に発現しており、 そのリガンドであるサイ トカインはしばしば造血因 子あるいはインターロイキンと称される。 これら造血因子、 あるいはインター ロイキン類のあるものは血流中に存在し全身的な造血あるいは免疫機能の体液 性調節に関与していると考えられる。 Representatives of the above cytokine family include tyrosine kinase receptor, hemopoietin receptor, tumor necrosis factor (TNF) receptor, and transforming growth factor /? (TGF?) Receptor. Family members, and the involvement of different signaling systems in each family has been reported. Of these receptor families—especially many of the hemopoietin receptor families are blood cells or immune Cytokine, which is expressed on the host cell and its ligand, is often called hematopoietic factor or interleukin. Some of these hematopoietic factors or interleukins are present in the bloodstream and may be involved in systemic hematopoiesis or humoral regulation of immune function.
このことは他のフアミ リーに対応するサイ トカインがしばしば局所での調節 にのみ関与していると考えられる点とは対照的で、 これらへモポェチン類の一 部のものはホルモン様因子と捉えることが可能であり、 また逆に代表的なぺプ チド性ホルモンである成長ホルモン、 プロラクチンあるいはレブチンの受容体 もへモポェチン受容体ファミリ一に属する。 上記ホルモン様の全身性調節様式 からこれらのへモポェチン類を投与することによる種々の疾患の治療への応用 が期待される。  This is in contrast to the fact that cytokines corresponding to other families are often thought to be involved only in local regulation, and some of these hemopoietins are regarded as hormone-like factors. Conversely, receptors for growth hormone, prolactin, or lebutin, which are typical peptide sex hormones, also belong to the hemopoietin receptor family. From the above-mentioned hormone-like systemic regulation mode, application of these hemopoetins to treatment of various diseases is expected.
事実、 数多いサイ ト力イン類の中で臨床応用が行われているのは、 エリス口 ポェチン、 G- CSF、 GM-CSF, IL-2であり、 また現在臨床応用に向けた検討が行わ れている、 IL- 11、 LIF、 IL-12に加えて上記ペプチドホルモン類の成長ホルモン、 プロラクチンを併せて考えると、 上記各種受容体フアミ リーのうちへモポェチ ン受容体に結合する新規サイ トカインを探索することにより、 より高い確率で 臨床応用可能なサイ トカインを見出すことが可能と考えられる。  In fact, among the many sites, clinical applications are being carried out on Eris Mouth Poetin, G-CSF, GM-CSF, IL-2, and are currently being studied for clinical applications. Considering the above-mentioned peptide hormones, growth hormone and prolactin, in addition to IL-11, LIF, and IL-12, a novel cytokine that binds to the hemopoietin receptor among the above various receptor families is considered. By searching, it is possible to find a clinically applicable cytokine that has a higher probability.
上に述べた様にサイ ト力ィン受容体はファミリーメンバ一間で構造上の類似 性を有している。 この類似性を利用して新規受容体を発見する試みは数多く行 われており、 特にチロシンキナーゼ受容体に関しては、 その触媒部位の高度に 保存された配列を利用して既に数多くの受容体がクローニングされている （Ma tthews W. et al . , Cell , 1991 , 65 ( 7) pll43- 52)。 これに対してへモポェチ ン受容体はその細胞質領域にチロシンキナーゼの様な酵素活性ドメインを有し ておらず、 そのシグナル伝達は細胞質中に遊離状態で存在する別のチロシンキ ナ一ゼ蛋白との会合を介して行われることが知られている。  As mentioned above, site force receptors have structural similarities among family members. Numerous attempts have been made to discover new receptors using this similarity, and particularly for tyrosine kinase receptors, many receptors have already been cloned using highly conserved sequences in their catalytic sites. (Matthews W. et al., Cell, 1991, 65 (7) pll43-52). In contrast, the hemopoietin receptor does not have an enzymatically active domain such as tyrosine kinase in its cytoplasmic region, and its signal transduction is associated with another tyrosine kinase protein that exists free in the cytoplasm. It is known to take place through a meeting.
これらの細胞質性チロシンキナーゼ（JAKキナーゼ） との受容体上の結合部位 はフアミリ一メンバ一間で一応保存されてはいるものの、 その相同性はあまり 高くない (Murakami M. et al.， Proc. Natl. Acad. Sci. USA, 1991， 88， 11349- 11353)。一方、 これらへモポェチン受容体を最もよく特徴付ける配列はむしろ 細胞外領域に存在し、 特に Trp-Ser-Xaa- Trp-Ser (Xaaは任意のアミノ酸） の 5 アミノ酸から成るモチーフは殆ど全てのへモポェチン受容体に保存されており、 この配列を利用した新規フアミリーメンバーの探索により新規受容体を取得す ることが期待される。 事実、 これまでに IL- 11受容体 （Robb, L. et al.， J. B iol. Chem. 271 (23)， 1996, 13754-13761) 、 レブチン受容体 (Gainsford T. et al., Proc.Natl.Acad. Sci. USA, 1996, 93 (25) pl4564- 8)及び IL- 13受容体 (Hilton. D.J. et al.， Proc.Natl.Acad. Sci. USA, 1996, 93 (1) p497-501) が このアプローチにより同定されている。 発明の開示 Binding sites on receptors for these cytoplasmic tyrosine kinases (JAK kinases) Although they are conserved between members of the family, their homology is not very high (Murakami M. et al., Proc. Natl. Acad. Sci. USA, 1991, 88, 11349-11353). On the other hand, the sequences that best characterize these hemopoietin receptors are rather present in the extracellular region, and in particular, the motif consisting of 5 amino acids of Trp-Ser-Xaa-Trp-Ser (Xaa is any amino acid) is almost all It is conserved in the receptor, and it is expected that a new receptor will be obtained by searching for a new family member using this sequence. In fact, IL-11 receptor (Robb, L. et al., J. Biol. Chem. 271 (23), 1996, 13754-13761), lebutin receptor (Gainsford T. et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (25) pl4564-8) and IL-13 receptor (Hilton. DJ et al., Proc. Natl. Acad. Sci. USA, 1996, 93 (1) p497 -501) has been identified by this approach. Disclosure of the invention
本発明は、 新規へモポェチン受容体蛋白質、 それをコードする DNAを提供 する。 本発明はまた、 該 DNAが挿入されたべクタ一、 該 DNAを保持する形 質転換体、 該形質転換体を利用した組換え蛋白質の製造方法を提供する。 本発 明はさらに、 該蛋白質に結合する化合物のスクリーニング方法を提供する。 本発明者らは、 これまでに、 Trp- Ser- Xaa-Trp-Serモチーフをコ一ドするオリ ゴヌクレオチドをプローブに用いてプラークハイブリダイゼ一シヨンあるいは RT-PCR等の方法により新規受容体の探索を試行してきた。 しかし、 このモチー フをコ一ドするオリゴヌクレオチド tggag(t/c)nnntggag(t/c)(nは任意の塩基） が 15塩基対と短いこと、さらには g/c含量が高い等の理由から通常のハイプリダ ィゼーシヨンの実験条件下で厳密に 15塩基が完全にハイプリダイズしたものだ けを選別することは極めて困難であった。  The present invention provides a novel hemopoietin receptor protein and a DNA encoding the same. The present invention also provides a vector into which the DNA has been inserted, a transformant retaining the DNA, and a method for producing a recombinant protein using the transformant. The present invention further provides a method for screening for a compound that binds to the protein. The present inventors have previously proposed a novel receptor by a method such as plaque hybridization or RT-PCR using an oligonucleotide coding for the Trp-Ser-Xaa-Trp-Ser motif as a probe. I have tried to search for However, the oligonucleotides encoding this motif, tggag (t / c) nnntggag (t / c) (where n is any base), are as short as 15 base pairs, and the g / c content is high. Therefore, it was extremely difficult to select only those whose strictly 15 bases were completely hybridized under the ordinary experimental conditions of hybridization.
また、 比較的広範に分布しかつ発現量も高いと考えられる各種コラーゲンを 初めとするへモポェチン受容体以外の蛋白をコードする c DN A中にも類似の 配列が含まれており、 これらの原因により上記プラークハイプリダイゼ一ショ ンあるいは RT-PCRによるスクリ一ニングは極めて効率の悪いものであつた。 これらの問題を解決するべく、 また実際ヒトゲノム上に幾つの異なるへモポ ェチン受容体遺伝子が存在するのかを推定する目的で、 上記 Trp- Ser- Xaa- Trp- Serモチーフをコードする全ての可能なォリゴヌクレオチド配列をプローブと してコンビユー夕上で個々のプローブと完全に一致する配列の検索を実施した 次に、 上記検索でヒッ トしたクローンのうちヒトゲノム由来クローン （コス ミ ド、 BAC、 PAC) について、 プローブ配列周辺の塩基配列をアミノ酸配列に変 換して既知のへモポェチン受容体のアミノ酸配列と比較することにより、 へモ ポェチン受容体フアミリ一メンバーをコードすると考えられるヒト遺伝子を選 別した。 Similar DNA is also present in cDNAs that encode proteins other than the hemopoietin receptor, including various collagens that are considered to be relatively widely distributed and highly expressed. For these reasons, screening by plaque hybridization or RT-PCR was extremely inefficient. In order to solve these problems and to estimate how many different hemopoietin receptor genes actually exist on the human genome, all possible encodings of the above Trp-Ser-Xaa-Trp-Ser motifs Using the oligonucleotide sequence as a probe, a search was performed for sequences that completely matched the individual probes on the combi- nation screen. Next, of the clones hit by the above search, clones derived from the human genome (cosmid, BAC, PAC), by converting the nucleotide sequence around the probe sequence to an amino acid sequence and comparing it with the amino acid sequence of a known hemopoietin receptor, a human gene thought to encode a member of the hemopoietin receptor familly was selected. Different.
以上の検索により 2つのへモポェチン受容体遺伝子と考えられるクローンを 同定した。 このうち 1つは既知の GM-CSFベ一夕受容体遺伝子 （第 22染色体 22ql 2.3- 13.2領域由来）であり、 もう 1つ （第 16染色体 16pl2領域由来 BACクローン A C002303)は新規へモポェチン受容体をコードすると推測され、 このヒ ト遺伝子 を 「冊 8」 と命名した。  Through the above search, two clones considered to be the hemopoietin receptor genes were identified. One of them is a known GM-CSF vector receptor gene (chromosome 22 from 22ql 2.3-13.2 region) and the other (chromosome 16 from 16pl2 region BAC clone A C002303) is a novel hemopoietin receptor. It was presumed to encode the body, and this human gene was named "Vol. 8".
次に、 得られた塩基配列をもとにしてデザィンされた特異的なプライマ一を 利用した RT-PCRにより NR8をコードすると考えられる c D N Aをヒト胎児肝細 胞 c D N Aライブラリ一中に見いだした。 さらに、 この c D N Aライブラリ一 を铸型に 5' - RACE法、 3' -RACE法を行うことにより最終的に 361アミノ酸からな る細胞膜貫通型受容体をコードする完全長 c D N A、 NR8ひを得た。  Next, a cDNA that is thought to encode NR8 was found in a human fetal liver cell cDNA library by RT-PCR using a specific primer designed based on the obtained nucleotide sequence. . Furthermore, by performing the 5'-RACE method and the 3'-RACE method using this cDNA library as a type I, a full-length cDNA encoding a 361 amino acid transmembrane receptor, NR8, was finally obtained. Obtained.
NR8ひは一次構造上、細胞外領域に他のフアミ リーメンバ一間で保存されてい るシスティン残基、 プロリンに富んだモチーフ、 細胞内領域にシグナル伝達に 関与すると考えられる boxlモチーフ等がよく保存されており典型的なへモポェ チン受容体と考えられた。  In the primary structure of NR8, cysteine residues conserved among other family members in the extracellular region, proline-rich motifs, and the boxl motif thought to be involved in signal transduction in the intracellular region are well conserved. And was considered a typical hemopoietin receptor.
さらに、 本発明者らは、 NR8ひの選択的スプライシング産物として、 それそれ M8 ?、 NR8ァと命名された 2つの遺伝子の存在を見出した。 In addition, the present inventors have proposed that as an alternative splicing product of NR8, We found the presence of two genes named M8? And NR8a.
本発明者等は、 次ぎに、 NR8遺伝子に対応するマウス遺伝子の単離を試みた。 まず、 ヒト NR8の cDNA配列内に設計した、ォリゴヌクレオチドプライマ一を利 用し、 異種間交叉 PCR クロ一ニングをマウス脳 cDNAライブラリ一を鎵型とし て実施することにより、 当該受容体のマウス部分塩基配列を単離した。 さらに 得られた部分配列をもとにオリゴヌクレオチドプライマ一を設計し、 それらを 用いた 5，- RACE法、及び 3'- RACE法を実施することで、 NR8に対応するマウス相 同遺伝子の全長 0RFの単離に成功した。得られた cDNAクローンの全塩基配列を 決定した結果、 NR8同様にスプライス変異体に由来する転写産物の相違により、 538アミノ酸からなる細胞膜貫通型受容体蛋白をコ一ドするマウス NR8ァと、 1 4 アミノ酸からなる分泌型の可溶性受容体様蛋白をコードするマウス NR8 ? の存在が確認された。 当該受容体遺伝子がコードするアミノ酸配列をヒトとマ ウス間において比較したところ、 NR8ァでは 98.9% の高い相同性が認められ、 一方 NR8 ?においても 97.2 の相同性を認めた。 さらに、 得られたマウス NR8 β cDNA 断片をプローブ として、 マウスゲノム DNA ライブラリーに対するプ ラークスクリ一ニングを実施することで、 目的の陽性クローンを単離すること に成功した。  Next, the present inventors tried to isolate a mouse gene corresponding to the NR8 gene. First, cross-species crossover PCR cloning is performed using a mouse brain cDNA library as a type I, using an oligonucleotide primer designed within the cDNA sequence of human NR8. The partial nucleotide sequence was isolated. Furthermore, oligonucleotide primers are designed based on the obtained partial sequences, and the 5, -RACE method and the 3'-RACE method using the primers are performed to obtain the entire length of the mouse homologous gene corresponding to NR8. 0RF was successfully isolated. As a result of determining the entire nucleotide sequence of the obtained cDNA clone, as in NR8, due to differences in transcripts derived from splice variants, mouse NR8a encoding a 538 amino acid transmembrane receptor protein, and 1 The presence of mouse NR8? Encoding a secretory soluble receptor-like protein consisting of 4 amino acids was confirmed. When the amino acid sequence encoded by the receptor gene was compared between human and mouse, a high homology of 98.9% was observed in NR8a, and a homology of 97.2 was also observed in NR8 ?. Furthermore, by performing plaque screening on the mouse genomic DNA library using the obtained mouse NR8β cDNA fragment as a probe, the target positive clone was successfully isolated.
従って、 本発明は、 （ 1 ) 配列番号： 1に示す 1位のアミノ酸 M e tから 3 6 1位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中 のアミノ酸配列において 1若しくは複数個のアミノ酸の欠失、 付加及び/又は 他のアミノ酸による置換により修飾されたアミノ酸配列からなり、 配列番号： 1に示す 1位のアミノ酸 M e tから 3 6 1位のアミノ酸 S e rまでのアミノ酸 配列からなる蛋白質と機能的に同等な蛋白質を提供する。  Accordingly, the present invention relates to (1) a protein consisting of the amino acid sequence from amino acid Me at position 1 to amino acid Ser at position 361 shown in SEQ ID NO: 1, or one or more amino acid sequences in the protein; Consisting of an amino acid sequence modified by the deletion, addition and / or substitution of another amino acid of the amino acid sequence, from the amino acid sequence from amino acid Met at position 1 to amino acid Ser at position 361 shown in SEQ ID NO: 1. To provide a protein functionally equivalent to the protein.
本発明はまた、 （2 ) 配列番号 ： 3に示す 1位のアミノ酸 M e tから 1 4 4 位のアミノ酸 L e uまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のァ ミノ酸配列において 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他の アミノ酸による置換により修飾されたアミノ酸配列からなり、 配列番号： 3に 示す 1位のアミノ酸 M e tから 1 4 4位のアミノ酸 L e uまでのアミノ酸配列 からなる蛋白質と機能的に同等な蛋白質を提供する。 The present invention also relates to (2) a protein consisting of the amino acid sequence from amino acid 1 at position 1 to amino acid Leu at position 144 shown in SEQ ID NO: 3, or one or more amino acid sequences in the amino acid sequence in the protein. Deletions, additions and / or other amino acids It provides a protein consisting of an amino acid sequence modified by substitution with an amino acid and functionally equivalent to a protein consisting of the amino acid sequence from the first amino acid Met to the 144th amino acid Leu shown in SEQ ID NO: 3. .
本発明はまた、 （3 ) 配列番号： 5に示す 1位のアミノ酸 M e tから 2 3 7 位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のァ ミノ酸配列において 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他の アミノ酸による置換により修飾されたアミノ酸配列からなり、 配列番号： 5に 示す 1位のアミノ酸 M e tから 2 3 7位のアミノ酸 S e rまでのアミノ酸配列 からなる蛋白質と機能的に同等な蛋白質を提供する。  The present invention also relates to (3) a protein consisting of the amino acid sequence from amino acid 1 at position 1 to amino acid Ser at position 237 shown in SEQ ID NO: 5, or one or more amino acid sequences in the amino acid sequence in the protein. Consisting of an amino acid sequence modified by deletion, addition and / or substitution of another amino acid, comprising the amino acid sequence from amino acid 1 at position 1 to amino acid Ser at position 237 shown in SEQ ID NO: 5. A protein functionally equivalent to a protein consisting of
本発明はまた、 （4 ) 配列番号： 7に示す 1位のアミノ酸 M e tから 5 3 8 位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のァ ミノ酸配列において 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他の アミノ酸による置換により修飾されたアミノ酸配列からなり、 配列番号： 7に 示す 1位のアミノ酸 M e tから 5 3 8位のアミノ酸 S e rまでのアミノ酸配列 からなる蛋白質と機能的に同等な蛋白質を提供する。  The present invention also relates to (4) a protein consisting of the amino acid sequence from amino acid 1 at position 1 to amino acid Ser at position 538 shown in SEQ ID NO: 7, or one or more amino acid sequences in the protein. Consisting of an amino acid sequence modified by deletion, addition and / or substitution of another amino acid, and comprising an amino acid sequence from the 1st amino acid Met shown in SEQ ID NO: 7 to the 538th amino acid Ser. A protein functionally equivalent to a protein consisting of
本発明はまた、 （ 5 ) 配列番号： 1 9示す 1位のアミノ酸 M e tから 1 4 4 位のアミノ酸 L e uまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のァ ミノ酸配列において 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他の アミノ酸による置換により修飾されたアミノ酸配列からなり、 配列番号： 1 9 に示す 1位のアミノ酸 M e tから 1 4 4位のアミノ酸 L e uまでのアミノ酸配 列からなる蛋白質と機能的に同等な蛋白質を提供する。  The present invention also relates to (5) a protein consisting of the amino acid sequence from amino acid Me at position 1 to amino acid Leu at position 144 shown in SEQ ID NO: 19, or one or more amino acid sequences in the amino acid sequence in the protein. Consisting of an amino acid sequence modified by deletion, addition, and / or substitution of another amino acid, the amino acids from the 1st amino acid Met shown in SEQ ID NO: 19 to the 144th amino acid Leu It provides a protein functionally equivalent to the protein consisting of the sequence.
本発明はまた、 （6 ) 配列番号： 2 1示す 1位のアミノ酸 M e から 5 3 8 位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のァ ミノ酸配列において 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他の アミノ酸による置換により修飾されたアミノ酸配列からなり、 配列番号： 2 1 に示す 1位のアミノ酸 M e tから 5 3 8位のアミノ酸 S e rまでのアミノ酸配 列からなる蛋白質と機能的に同等な蛋白質を提供する。 The present invention also relates to (6) a protein consisting of an amino acid sequence from amino acid Me at position 1 to amino acid Ser at position 538 shown in SEQ ID NO: 21 or one or more amino acid sequences in the amino acid sequence in the protein. Consisting of an amino acid sequence modified by deletion, addition and / or substitution of another amino acid, the amino acids from amino acid Met at position 1 shown in SEQ ID NO: 21 to amino acid Ser at position 538 Arrangement Provides a protein functionally equivalent to the protein in the sequence.
本発明はまた、 （7) 配列番号： 2に記載の塩基配列からなる DNAとハイ ブリダィズする DNAがコードする蛋白質であって、 配列番号： 1に示す 1位 のアミノ酸 Me tから 361位のアミノ酸 Serまでのアミノ酸配列からなる 蛋白質と機能的に同等な蛋白質を提供する。  The present invention also provides (7) a protein encoded by a DNA that hybridizes with the DNA consisting of the nucleotide sequence of SEQ ID NO: 2, wherein the amino acid at position 1 to the amino acid at position 361 to the amino acid at position 361 shown in SEQ ID NO: 1 Provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to Ser.
本発明はまた、 （8) 配列番号： 4に記載の塩基配列からなる DNAとハイ ブリダィズする DNAがコードする蛋白質であって、 配列番号： 3に示す 1位 のアミノ酸 Me tから 144位のアミノ酸 L e uまでのアミノ酸配列からなる 蛋白質と機能的に同等な蛋白質を提供する。  The present invention also provides (8) a protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 4, wherein the amino acid at position 1 to the amino acid at position 144 to the amino acid at position 144 shown in SEQ ID NO: 3 It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to Leu.
本発明はまた、 （9) 配列番号： 6に記載の塩基配列からなる DNAとハイ ブリダィズする DNAがコードする蛋白質であって、 配列番号： 5に示す 1位 のアミノ酸 Me tから 237位のアミノ酸 S e rまでのアミノ酸配列からなる 蛋白質と機能的に同等な蛋白質を提供する。  The present invention also provides (9) a protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 6, wherein the amino acid at position 1 to the amino acid at position 237 shown in SEQ ID NO: 5 It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to Ser.
本発明はまた、 （10) 配列番号： 8に記載の塩基配列からなる DN Aとハ イブリダィズする DNAがコードする蛋白質であって、 配列番号： 7に示す 1 位のアミノ酸 Me tから 538位のアミノ酸 S e rまでのアミノ酸配列からな る蛋白質と機能的に同等な蛋白質を提供する。  The present invention also relates to (10) a protein encoded by a DNA hybridizing with the DNA comprising the nucleotide sequence of SEQ ID NO: 8; It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to the amino acid Ser.
本発明はまた、 （11) 配列番号： 20に記載の塩基配列からなる DN Aと ハイブリダィズする DN Aがコードする蛋白質であって、 配列番号： 19に示 す 1位のアミノ酸 Me tから 144位のアミノ酸 Leuまでのアミノ酸配列か らなる蛋白質と機能的に同等な蛋白質を提供する。  The present invention also relates to (11) a protein encoded by a DNA which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 20; It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to amino acid Leu.
本発明はまた、 （12) 配列番号： 22に記載の塩基配列からなる DN A とハイブリダィズする DNAがコードする蛋白質であって、 配列番号： 21に 示す 1位のアミノ酸 Me tから 538位のアミノ酸 S e rまでのアミノ酸配列 からなる蛋白質と機能的に同等な蛋白質を提供する。  The present invention also relates to (12) a protein encoded by a DNA hybridizing with DNA comprising the nucleotide sequence of SEQ ID NO: 22, wherein the amino acid at the 1st position and the amino acid at the 538th position are shown in SEQ ID NO: 21. It provides a protein functionally equivalent to a protein consisting of the amino acid sequence up to Ser.
本発明はまた、 （ 13) 上記 （ 1) 〜 （ 12) のいずれか 1つに記載の蛋白 質と他のぺプチド又はポリベプチドとからなる融合蛋白質を提供する。 (13) The protein according to any one of (1) to (12) above, A fusion protein comprising a protein and another peptide or polypeptide is provided.
本発明はまた、 （ 14) 上記 （ 1 ) ~ ( 13) のいずれか 1つに記載の蛋白 質をコードする DN Aを提供する。  The present invention also provides (14) a DNA encoding the protein described in any one of (1) to (13) above.
本発明はまた、 （15) 上記 （ 14) に記載の DNAが挿入されたべクタ一 を提供する。  The present invention also provides (15) a vector into which the DNA of (14) has been inserted.
本発明はまた、 （16) 上記 （ 14) に記載の DNAを発現可能に保持する 形質転換体を提供する。  The present invention also provides (16) a transformant capable of expressing the DNA of (14) above.
本発明はまた、 （17) 上記 （ 16) に記載の形質転換体を培養する工程を 含む、 上記 （ 1 ) 〜 （ 13 ) のいずれか 1つに記載の蛋白質の製造方法を提供 する。  The present invention also provides (17) a method for producing the protein according to any one of the above (1) to (13), comprising a step of culturing the transformant according to the above (16).
本発明はまた、 （ 18) 上記 （ 1) 〜 （ 13) に記載の蛋白質に結合する化 合物をスクリーニングする方法であって、  The present invention also provides (18) a method for screening a compound that binds to the protein according to any of (1) to (13) above,
(a) 上記 （ 1) 〜 （ 13) のいずれか 1つに記載の蛋白質に被験試料を接触 させる工程、 および  (a) contacting a test sample with the protein according to any one of the above (1) to (13), and
(b) 上記 （ 1) 〜 （13) のいずれか 1つに記載の蛋白質に結合する活性を 有する化合物を選択する工程、 を含む方法を提供する。  (b) selecting a compound having an activity of binding to the protein according to any one of the above (1) to (13).
本発明はまた、 （ 19 ) 上記 （ 1 ) 〜 （ 12) のいずれか 1つに記載の蛋白 質に対して特異的に結合する抗体を提供する。  The present invention also provides (19) an antibody that specifically binds to the protein according to any one of (1) to (12).
本発明はまた、 （20)上記 （ 19 ) に記載の抗体と、 上記 （ 1 )〜 （ 13) のいずれか 1つに記載の蛋白質が含まれると予想される試料とを接触せしめ、 前記抗体と該蛋白質との免疫複合体の生成を検出又は測定することを含んでな る該蛋白質の検出又は測定方法を提供する。  The present invention also provides (20) contacting the antibody according to (19) with a sample expected to contain the protein according to any one of (1) to (13), A method for detecting or measuring the production of an immunocomplex comprising the protein and a protein.
本発明はまた、 （21) 配列番号： 2、 4、 6、 8、 20、 および 22から 27のいずれか一つに記載の塩基配列からなる DN Aと特異的にハイプリダイ ズし、 少なくとも 15塩基の鎖長を有する DNAを提供する。  The present invention also provides (21) a DNA which specifically hybridizes with a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 2, 4, 6, 8, 20, and 22 to 27, and has at least 15 nucleotides. DNA having a chain length of
本発明は、 新規へモポェチン受容体 「NR8」 に関する。 本発明者らによる、 5 RACE及び 3' -RACEによる解析、 NR8ゲノム配列の解析、 さらにはプラークスク リーニングによる解析の結果から、 NR8ひ、 NR8 、 NR8ァの存在が予測された。 これら NR8遺伝子の構造を図 1 3に示す。 NR8遺伝子のうち、 NR8 3は第 5ェクソ ンを欠いたオル夕ナティブスプライス産物であり、 第 4ェクソンに直結しフレ 一ムシフ 卜を起こして生じた第 6ェクソン上の終止コドンで CDSが終結する可 溶性蛋白質と第 4ェクソン上の ATGから開始するシグナル配列を欠いた膜結合 蛋白の 2種の異なる蛋白をコードすることが可能である。 The present invention relates to a novel hemopoietin receptor "NR8". According to the present inventors, 5 The results of analysis by RACE and 3'-RACE, analysis of NR8 genome sequence, and analysis by plaque screening predicted the presence of NR8, NR8, and NR8a. The structures of these NR8 genes are shown in FIG. Of the NR8 genes, NR83 is an alternative splice product lacking the fifth exon, and CDS terminates at the stop codon on the sixth exon that is directly linked to the fourth exon and causes a frame shift. It is possible to encode two different proteins, a soluble protein that binds to a protein and a membrane-bound protein that lacks a signal sequence starting from ATG on the fourth exon.
このうち可溶性蛋白は NR8ひと第 4ェクソンまでは同じアミノ酸配列をとる ことから可溶性受容体として機能する可能性がある。一方、 NR8ァはやはり選択 的スプライシングの結果、 NR8ひの C末端近傍に NR8第 9ィントロンに由来する 1 77アミノ酸の挿入を含む蛋白質をコードする。  Among them, the soluble protein has the same amino acid sequence up to the fourth exon of NR8 and may function as a soluble receptor. On the other hand, NR8a also encodes a protein containing an insertion of 177 amino acids from the NR8 intron 9 near the C-terminus of NR8 as a result of alternative splicing.
NR8ひと NR8ァはともに細胞膜貫通型のへモポェチン受容体をコードする。 NR 8ひ及び NR8ァの細胞内ドメインにはシグナル伝達に関与すると考えられる他の へモポェチン受容体間で保存されている配列のうち Boxlに似たモチーフが細胞 膜近傍に存在する。 Box2についても保存度は低いながら類似の配列が存在する ことから、 NR8はホモ 2量体でシグナルを伝達するタイプの受容体に属するもの と考えられる。  Both NR8 and NR8 encode a transmembrane hemopoietin receptor. In the intracellular domain of NR8 and NR8a, a motif similar to Box1 exists in the vicinity of the cell membrane among sequences conserved among other hemopoietin receptors that are considered to be involved in signal transduction. Since similar sequences exist with low conservation in Box2, NR8 is considered to belong to the type of receptor that transmits signals as homodimers.
本発明の蛋白質に含まれる NR8と命名された蛋白質のアミノ酸配列を配列番 号： 1 (NR8ひ） 、 配列番号： 3 (可溶型 NR8 5 ) 、 配列番号： 5 (膜結合型 NR S/3 ) 及び配列番号： Ί (NR8ァ） に示し、 該蛋白質をコードする c D N Aの塩 基配列を配列番号： 2、 配列番号： 4、 配列番号： 6及び配列番号： 8に示す。 また、 ノーザン解析の結果、 脾臓、 胸腺、 末梢白血球、 肺において、 5kbと 3 -4kbの領域に 2- 3本のバンドを認めた。細胞株についても同様なサイズのバンド を HL60と Rajiに認め、 他の癌細胞株 （HeLa、 SW480, A549、 G361 ) 及び白血病細 胞株 （K562、 M0LT4) には全く発現を認めなかった。  The amino acid sequence of the protein designated as NR8 contained in the protein of the present invention is represented by SEQ ID NO: 1 (NR8), SEQ ID NO: 3 (soluble NR85), SEQ ID NO: 5 (membrane-bound NR S / 3) and SEQ ID NO: Ί (NR8a), and the nucleotide sequences of the cDNA encoding the protein are shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. As a result of Northern analysis, 2-3 bands were observed in the 5 kb and 3 -4 kb regions in the spleen, thymus, peripheral leukocytes, and lung. In the cell lines, bands of similar size were observed in HL60 and Raji, and no expression was observed in other cancer cell lines (HeLa, SW480, A549, G361) and leukemia cell lines (K562, M0LT4).
以上の結果は NR8が造血細胞系、 特に顆粒球系と Β細胞系に特異的に発現して いることを示唆する。 These results indicate that NR8 is specifically expressed in hematopoietic cell lines, especially in granulocyte and Β cell lines. Implies that
上記 NR8蛋白質には、 医療への応用が考えられる。 NR8が胎児肝、 脾臓、 胸腺 及びある種の白血病細胞株に発現していることから未知の造血因子の受容体で ある可能性が示唆される。従って、 NR8蛋白はこの未知の造血因子を得るための 有用な材料を提供するものと考えられる。  The NR8 protein described above has potential medical applications. Expression of NR8 in fetal liver, spleen, thymus and certain leukemia cell lines suggests that it may be a receptor for unknown hematopoietic factors. Therefore, NR8 protein is considered to provide a useful material for obtaining this unknown hematopoietic factor.
また、 NR8の発現はこれら造血組織中の限られた細胞集団に特異的に発現して いる可能性が想定され、この細胞集団を分離する手段として抗 NR8抗体は有用で ある。この様にして分離された細胞集団は細胞移植療法への応用が可能である。 さらに抗 NR8抗体は白血病を初めとした疾患の病型診断あるいは治療への応用 も期待される。  In addition, it is assumed that NR8 expression may be specifically expressed in a limited cell population in these hematopoietic tissues, and an anti-NR8 antibody is useful as a means for separating this cell population. The cell population thus separated can be applied to cell transplantation therapy. Furthermore, anti-NR8 antibodies are also expected to be applied to the diagnosis or treatment of leukemia and other diseases.
一方、 NR8蛋白質の細胞外ドメインを含む可溶性蛋白質、 あるいは NR8のスプ ライス変異体である NR8 ?は decoy型受容体として NR8リガンドの阻害剤として の利用が想定され、 NR8が関与する白血病を初めとする疾患の治療への応用が期 待できる。  On the other hand, soluble proteins containing the extracellular domain of NR8 protein, or NR8 splice variant NR8 ?, are expected to be used as decoy-type receptors as inhibitors of NR8 ligands, including leukemia involving NR8. It can be expected to be applied to the treatment of illness.
また、 本発明者等は、 異種間交叉 PCR クローニングの手法を利用して、 上記 ヒト由来の NR8 cDNAに対応するマウス NR8 cDNAを単離した。 本発明の蛋白質に 含まれるマウス NR8と命名された蛋白質のアミノ酸配列を配列番号： 1 9 (可溶 型マウス NR8 ? ) と配列番号： 2 1 (マウス NR8ァ） に示し、 該蛋白質をコード する c D N Aの塩基配列をそれそれ配列番号： 2 0と配列番号： 2 2に示す。 得られたマウス cDNAクローンの構造解析の結果、ヒ ト由来の NR8同様にスプ ライス変異体に由来する転写産物の相違により、 538 アミノ酸からなる細胞膜 貫通型受容体蛋白をコ一ドするマウス NR8ァと、 144ァミノ酸からなる分泌型の 可溶性受容体様蛋白をコードするマウス NR8 5の存在が確認された。 当該受容 体遺伝子がコードするアミノ酸配列をヒ トとマウス間において比較したところ、 NR8ァでは 98.9% の高い相同性が認められ、一方 においても 97.2 の相同 性を認めた。 ノーザン解析および RT- PCR解析により、 マウス NR8遺伝子は、発現量に偏差 はあったものの、 解析した全ての臓器で発現が認められるなど、 免疫担当組織 や造血組織においてのみ強い発現が認められたヒト由来の NR8に対し、 その遺 伝子発現は広範な存在分布を示した。 これは、 マウスにおける NR8の分子機能 が、 多岐にわたる生体の生理調節機構に関与する可能性をも示唆している。 本発明はまた、上記ヒ トおよびマウス NR8蛋白質と機能的に同等な蛋白質も包 含する。 本発明において 「機能的に同等」 とは、 蛋白質が、 上記 NR8蛋白質と同 等の生物学的活性を有することを指す。 生物学的活性としては、 例えば、 造血 因子受容体蛋白質活性である。 このような蛋白質を得るための方法としては、 蛋白質のアミノ酸配列に変異を導入する方法が用いられている。 例えば、 合成 オリゴヌクレオチドプライマ一を利用した部位特異的変異誘発法により、 蛋白 質中のアミノ酸配列に所望の変異を導入することができる （Kramer, W. and F ritz, H. J. Methods in Enzymol . ( 1987) 154， 350-367) 。 また、 PCRによる 部位特異的変異誘発システム （GIBCO- BRL製） を使用して、 蛋白質中のアミノ酸 配列に変異を導入することもできる。 これらの方法により、 配列番号： 1、 配 列番号： 3、 配列番号： 5、 配列番号： 7、 配列番号： 1 9、 または配列番号： 2 1に示されたアミノ酸配列からなる蛋白質において、 その生物学的活性に影 響を与えないよう、 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他の アミノ酸による置換により修飾された、 NR8蛋白質と機能的に同等な蛋白質を得 ることができる。 Further, the present inventors have isolated a mouse NR8 cDNA corresponding to the above-mentioned human-derived NR8 cDNA by using the technique of cross-species cross-PCR cloning. The amino acid sequence of the protein designated as mouse NR8 contained in the protein of the present invention is shown in SEQ ID NO: 19 (soluble mouse NR8?) And SEQ ID NO: 21 (mouse NR8a), and encodes the protein. The nucleotide sequences of the cDNA are shown in SEQ ID NO: 20 and SEQ ID NO: 22, respectively. As a result of structural analysis of the obtained mouse cDNA clone, mouse NR8 gene encoding a 538 amino acid transmembrane receptor protein was identified due to differences in the transcripts derived from the splice variant as well as human NR8. Thus, the presence of mouse NR85 encoding a secreted soluble receptor-like protein consisting of 144 amino acids was confirmed. When the amino acid sequence encoded by the receptor gene was compared between human and mouse, a high homology of 98.9% was observed in NR8a, and a homology of 97.2 was also observed in NR8a. Although the expression level of mouse NR8 gene varied by Northern analysis and RT-PCR analysis, expression was observed in all analyzed organs. For NR8 of origin, its gene expression showed a wide distribution. This suggests that the molecular function of NR8 in mice may be involved in a wide variety of biological physiological regulatory mechanisms. The present invention also encompasses proteins functionally equivalent to the above-mentioned human and mouse NR8 proteins. In the present invention, “functionally equivalent” means that the protein has the same biological activity as the NR8 protein. The biological activity is, for example, hematopoietic factor receptor protein activity. As a method for obtaining such a protein, a method of introducing a mutation into the amino acid sequence of the protein has been used. For example, a desired mutation can be introduced into an amino acid sequence in a protein by site-directed mutagenesis using a synthetic oligonucleotide primer (Kramer, W. and Fritz, HJ Methods in Enzymol. (1987). ) 154, 350-367). In addition, mutations can be introduced into the amino acid sequence in a protein using a site-directed mutagenesis system (manufactured by GIBCO-BRL) by PCR. According to these methods, a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 19, or SEQ ID NO: 21 It is possible to obtain a protein functionally equivalent to the NR8 protein, modified by deletion or addition of one or more amino acids and / or substitution with another amino acid so as not to affect the biological activity. it can.
本発明の NR8蛋白質と機能的に同等な蛋白質としては、 具体的には、 配列番 号： 1、 配列番号 ： 3、 配列番号： 5、 配列番号 ： 7、 配列番号： 1 9、 およ び配列番号： 2 1のいずれかに示されるアミノ酸配列中の 1又は 2個以上、 好 ましくは、 2個以上 3 0個以下、 より好ましくは 2個以上 1 0個以下のァミノ 酸が欠失したもの、 配列番号： 1、 配列番号： 3、 配列番号： 5及び配列番号： 7のいずれかに示されるアミノ酸配列に 1又は 2個以上、 好ましくは、 2個以 上 3 0個以下、より好ましくは 2個以上 1 0個以下のァミノ酸が付加したもの、 配列番号： 1、 配列番号： 3、 配列番号 ： 5及び配列番号： 7のいずれかに示 されるアミノ酸配列中の 1又は 2個以上、 好ましくは、 2個以上 3 0個以下、 より好ましくは 2個以上 1 0個以下のアミノ酸が他のアミノ酸で置換されたも のが挙げられる。 Specific examples of the protein functionally equivalent to the NR8 protein of the present invention include SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 19, and SEQ ID NO: 21 One or more, preferably two or more and 30 or less, more preferably two or more and 10 or less amino acids in the amino acid sequence shown in any of SEQ ID NO: 21 are deleted. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7, one or more, preferably two or more SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7 in which 30 or less, more preferably 2 or more and 10 or less amino acids are added. One or more amino acids in the amino acid sequence, preferably two or more and 30 or less, more preferably two or more and 10 or less amino acids are substituted with other amino acids.
あるアミノ酸配列に対する 1又は複数個のアミノ酸残基の欠失、 付加及び/ 又は他のアミノ酸による置換により修飾されたアミノ酸配列を有する蛋白質が その生物学的活性を維持することはすでに知られている（Mark, D. F. et al .， Proc . Natl . Acad. Sci . USA ( 1984) 81 , 5662-5666 、 Zol ler, M. J. & Smi th, . Nucleic Acids Research ( 1982 ) 10, 6487-6500 、 Wang, A. et al .， Science 224, 1431-1433 、 Dalbadie-McFarland, G. et al .， Proc. Natl . A cad. Sci . USA ( 1982) 79, 6409-6413) 。  It is already known that a protein having an amino acid sequence modified by deleting, adding, and / or substituting one or more amino acid residues for a certain amino acid residue maintains its biological activity. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, MJ & Smith,. Nucleic Acids Research (1982) 10, 6487-6500, Wang, A (Mark, DF et al., Proc. Natl. Acad. Sci. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).
例えば、本発明の NR8蛋白質に 1又は複数個のアミノ酸残基が付加された蛋白 質として、 融合蛋白質が挙げられる。融合蛋白質は、 本発明の NR8蛋白質と他の ペプチド又は蛋白質とが融合したものであり、 本発明に包含される。 融合蛋白 質を作製する方法は、本発明の NR8蛋白質をコ一ドする D N Aと他のぺプチド又 は蛋白質をコ一ドする D N Aをフレームが一致するように連結してこれを発現 ベクターに導入し、 宿主で発現させればよく、 すでに公知の手法を用いること ができる。 本発明の蛋白質との融合に付される他のぺプチド又は蛋白質として は、 特に限定されない。  For example, a fusion protein may be mentioned as a protein obtained by adding one or more amino acid residues to the NR8 protein of the present invention. The fusion protein is a fusion of the NR8 protein of the present invention and another peptide or protein, and is included in the present invention. The fusion protein is prepared by ligating the DNA encoding the NR8 protein of the present invention and the DNA encoding the other peptide or protein so that their frames match, and introducing this into an expression vector. Alternatively, the expression may be carried out in a host, and a known method can be used. Other peptides or proteins to be fused with the protein of the present invention are not particularly limited.
例えば、 ペプチドとしては、 FLAG (Hopp, T. P. et al . , BioTechnology ( 1 988) 6, 1204-1210) 、 6個の His (ヒスチジン） 残基からなる 6 xHis、 lO xHis, インフルエンザ凝集素 （HA) 、 ヒト c-myc の断片、 VSV-GPの断片、 pl8HIVの断 片、 T7-tag、 HSV-tag, E-tag、 SV40T 抗原の断片、 lck tag 、 ひ- tubul inの断 片、 B-tag、 Protein C の断片等、 すでに公知であるべプチドが使用される。 ま た蛋白質としては、 例えば GST (グル夕チオン トランスフェラーゼ） 、 HA (インフルエンザ凝集素） 、 ィムノグロブリン定常領域、 3—ガラクトシダ一 ゼ、 MBP (マルト一ス結合蛋白質） 等が挙げられる。市販されているこれらをコ 一ドする DN Aを融合させたものを用いることができる。 For example, peptides include FLAG (Hopp, TP et al., BioTechnology (1 988) 6, 1204-1210), 6 x His consisting of 6 His (histidine) residues, lO x His, influenza agglutinin (HA) , Human c-myc fragment, VSV-GP fragment, pl8HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck tag, single-tubulin fragment, B-tag A known peptide such as a fragment of Protein C is used. As proteins, for example, GST (glucathione transferase), HA (Influenza agglutinin), immunoglobulin constant region, 3-galactosidase, MBP (maltose binding protein) and the like. A commercially available product obtained by fusing DNA encoding these can be used.
本発明の蛋白質はまた、 配列番号： 2、 配列番号： 4、 配列番号： 6、 配列 番号： 8、 配列番号： 20および配列番号： 22 ら 27のいずれかに示され る塩基配列からなる DNAとストリンジェン卜な条件下でハイプリダイズする DNAによりコ一ドされており、且つ上記 NR8蛋白質と機能的に同等な蛋白質を 含む。 ストリンジヱン卜な条件としては、 当業者であれば適宜選択することが できる力 例えば低ストリンジェントな条件が挙げられる。 低ストリンジェン 卜の条件とは、 例えば 42°C、 2xSSC、 0.1%SDSが挙げられ、 好ましくは 50°C、 2 xSSC、 0.1%SDSである。 またより好ましくは、 高ストリンジェン卜な条件が挙 げられる。 高ストリンジェン卜な条件とは、 例えば 65°C、 2XSSC及び 0.1%SDS が挙げられる。 これらの条件において、 温度を上げる程に高い相同性を有する DN Aを得ることができる。  The protein of the present invention also comprises a DNA consisting of the nucleotide sequence shown in any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20, and SEQ ID NOS: 22 to 27. And a protein that is encoded by DNA that hybridizes under stringent conditions and that is functionally equivalent to the NR8 protein. Stringent conditions include those that can be appropriately selected by those skilled in the art, for example, low stringency conditions. The conditions of low stringency include, for example, 42 ° C., 2 × SSC, 0.1% SDS, and preferably 50 ° C., 2 × SSC, 0.1% SDS. More preferably, high stringency conditions can be mentioned. High stringency conditions include, for example, 65 ° C, 2XSSC and 0.1% SDS. Under these conditions, DNA with higher homology can be obtained as the temperature is increased.
また、 本発明には上記 NR8蛋白質と機能的に同等であり、 且つ配列番号： 1、 配列番号： 3、 配列番号： 5、 配列番号： 7、 配列番号： 1 9および配列番号： 2 1のいずれかに示されるアミノ酸配列を有する蛋白質と相同性を有する蛋白 質も含まれる。 相同性を有する蛋白質とは、 配列番号： 1、 配列番号： 3、 配 列番号： 5及び配列番号： 7のいずれかに示されるアミノ酸配列と、 少なくと も 70%、 好ましくは少なくとも 80%、 より好ましくは少なくとも 90%、 さらに 好ましくは少なくとも 95%以上、 ァミノ酸配列上の相同性を有する蛋白質を意 味する。 蛋白質の相同性を決定するには、 文献 （Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80， 726-730)に記載のァルゴリズ ムにしたがえばよい。  Also, in the present invention, SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 19 and SEQ ID NO: 21 are functionally equivalent to the above-mentioned NR8 protein. Also included are proteins having homology to a protein having any of the amino acid sequences shown in any of them. The protein having homology is defined as at least 70%, preferably at least 80%, of the amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. More preferably, it means a protein having at least 90%, more preferably at least 95% or more homology on the amino acid sequence. To determine the homology of proteins, the algorithm described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730) may be used.
本発明の蛋白質は、 後述するそれを産生する細胞や宿主あるいは精製方法に より、 アミノ酸配列、 分子量、 等電点又は糖鎖の有無や形態が異なっている。 しかしながら、得られた蛋白質が造血因子受容体蛋白質活性を有している限り、 本発明に含まれる。 The protein of the present invention differs in the amino acid sequence, molecular weight, isoelectric point, presence / absence and form of a sugar chain depending on the cell, host, or purification method that produces the protein as described below. However, as long as the obtained protein has hematopoietic factor receptor protein activity, it is included in the present invention.
例えば、 本発明の蛋白質を原核細胞、 例えば大腸菌で発現させた場合、 本来 の蛋白質のアミノ酸配列の N末端にメチォニン残基が付加される。また、真核細 胞、例えば哺乳動物細胞で発現させた場合、 N末端のシグナル配列は除去される。 本発明の蛋白質はこのような蛋白質も包含する。  For example, when the protein of the present invention is expressed in a prokaryotic cell, for example, Escherichia coli, a methionine residue is added to the N-terminal of the amino acid sequence of the original protein. When expressed in eukaryotic cells, for example, mammalian cells, the N-terminal signal sequence is removed. The proteins of the present invention also include such proteins.
例えば、 文献 ( Von Heijne, G. Nucleic Acids Research ( 1986 ) 14, 4683- 4690) に記載の方法に基づいて、 本発明の蛋白質を解析した結果、 シグナル配 列は配列番号： 1のアミノ酸配列において、 1位の Metから 19位の Glyまでと推定 された。 したがって、 本発明は配列番号： 1に記載のアミノ酸配列において、 2 0位の Cysから 361位の Serまでからなる蛋白質を包含する。  For example, as a result of analyzing the protein of the present invention based on the method described in the literature (Von Heijne, G. Nucleic Acids Research (1986) 14, 4683-4690), the signal sequence was determined to be the amino acid sequence of SEQ ID NO: 1 It was estimated that Met from 1st place to Gly in 19th place Accordingly, the present invention includes a protein consisting of Cys at position 20 to Ser at position 361 in the amino acid sequence of SEQ ID NO: 1.
本発明の蛋白質を製造するには、 得られた D N Aを発現制御領域、 例えばェ ンハンサー、 プロモー夕一の制御のもとで発現可能なように発現ベクターに組 み込む。 次に、 この発現ベクターにより宿主細胞を形質転換し、 蛋白質を発現 させる。  To produce the protein of the present invention, the obtained DNA is incorporated into an expression vector so that it can be expressed under the control of an expression control region, for example, an enhancer or promoter. Next, a host cell is transformed with this expression vector to express the protein.
具体的には次のようにすればよい。 哺乳類細胞を使用する場合、 常用される 有用なプロモ一夕一/ェンハンサ一、 本発明の蛋白質をコードする D N A、 そ の 3'側下流にポリ A シグナルを機能的に結合させた D N Aあるいはそれを含む ベクターを構築する。 例えばプロモー夕一/ェンハンサ一としては、 ヒトサイ トメガロウィルス前期プロモ一夕一/ェンハンサ一 (human cytomegalovirus immediate early promoter/enhancer) を挙げることができる。  Specifically, the following may be performed. When using mammalian cells, useful promoters / enhancers commonly used, DNA encoding the protein of the present invention, DNA having a polyA signal functionally linked to its 3 ′ downstream, or DNA Construct a vector containing For example, the promoter / enhancer can be a human cytomegalovirus immediate early promoter / enhancer.
また、その他に蛋白質発現に使用できるプロモー夕—ノエンハンサ一として、 レトロウイルス、 ポリオ一マウィルス、 アデノウイルス、 シミアンウィルス 40 ( SV 40)等のウィルスプロモ一夕一/ェンハンサ一ゃヒトェロンゲ一シヨンフ アクター 1ひ (HEFl a )の哺乳類細胞由来のプロモ一夕一/ェンハンサ一を用い ればよい。 例えば、 SV 40プロモー夕一/ェンハンサーを使用する場合、 Mulliganらの方 法 （Nature ( 1979 ) 277, 108) 、 また、 HEF1ひプロモー夕一/ェンハンサ一を 使用する場合、 Mizushimaらの方法 (Nucleic Acids Res . ( 1990 ) 18， 5322) に 従えば容易に実施することができる。 Other promoters that can be used for protein expression include retroviruses, polioviruses, adenoviruses, Simian virus 40 (SV40), and other virus promoters. The promoter (HEFla) derived from mammalian cells may be used. For example, the method of Mulligan et al. (Nature (1979) 277, 108) when using the SV 40 Promoter / Enhancer, and the method of Mizushima et al. (Nucleic (1990) 18, 5322).
大腸菌を使用する場合、 常用される有用なプロモーター、 ポリペプチド分泌 のためのシグナル配列、 発現させる遺伝子を機能的に結合させて発現させるこ とができる。 例えばプロモータ一としては、 lacZプロモ一夕一、 araBプロモー 夕一を挙げることができる。 lacZプロモータ一を使用する場合、 Wardらの方法 (Nature ( 1098 ) 341， 544-546； FASEB J . ( 1992 ) 6， 2422-2427) 、 araBプロ モーターを使用する場合、 Betterらの方法 （Science ( 1988) 240, 1041-1043) に従えばよい。  When Escherichia coli is used, expression can be performed by operably linking a useful promoter commonly used, a signal sequence for polypeptide secretion, and a gene to be expressed. For example, the promoters include the lacZ promoter and the araB promoter. When the lacZ promoter is used, the method of Ward et al. (Nature (1098) 341, 544-546; FASEB J. (1992) 6, 2422-2427), and when the araB promoter is used, the method of Better et al. (Science (1988) 240, 1041-1043).
蛋白質分泌のためのシグナル配列としては、 大腸菌のペリブラズムに産生さ せる場合、 pelBシグナル配列 (Lei , S. P . et al J. Bacteriol . ( 1987) 169， 4379) を使用すればよい。  As a signal sequence for protein secretion, a pelB signal sequence (Lei, SP. Et al J. Bacteriol. (1987) 169, 4379) may be used when produced by E. coli periplasm.
複製開始点としては、 SV 40、 ポリオ一マウィルス、 アデノウイルス、 ゥシパ ピロ一マウィルス （BPV) 等の由来のものを用いることができる。 さらに、 宿主 細胞系で遺伝子コピー数増幅のため、 発現べクタ一は選択マーカーとして、 ァ ミノグリコシド トランスフェラ一ゼ （APH) 遺伝子、 チミジンキナーゼ （TK)遺 伝子、大腸菌キサンチングァニンホスホリボシルトランスフェラ一ゼ（Ecogpt) 遺伝子、 ジヒドロ葉酸還元酵素 （dhfr) 遺伝子等を含むことができる。  As the origin of replication, those derived from SV40, poliovirus, adenovirus, パ papilloma virus (BPV) and the like can be used. In addition, the expression vector can be used as a selection marker to amplify the gene copy number in the host cell system, such as the aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, and Escherichia coli xanthinguanine phosphoribosyltrans. It can contain the ferragase (Ecogpt) gene, the dihydrofolate reductase (dhfr) gene, and the like.
本発明の蛋白質を製造するための発現べクタ一は、 本発明に好適に使用され る発現ベクターであればいかなる発現べクタ一であってよい。 本発明の発現べ クタ一としては、 哺乳動物由来の発現ベクター、 例えば pEF、 pCDM8、 昆虫細胞 由来の発現べクタ一、 例えば pBacPAK8、 植物由来の発現ベクター、 例えば ρΜΗ1、 pMH2、 動物ウィルス由来の発現べクタ一、 例えば pHSV、 pMV、 pAdexLcw, レトロ ウィルス由来の発現ベクター、 例えば pZIpneo、 酵母由来の発現ベクター、 例え lb The expression vector for producing the protein of the present invention may be any expression vector as long as it is an expression vector suitably used in the present invention. Examples of the expression vector of the present invention include expression vectors derived from mammals, for example, pEF, pCDM8, expression vectors derived from insect cells, for example, pBacPAK8, expression vectors derived from plants, such as ρΜΗ1, pMH2, and expression derived from animal viruses. Vectors, such as pHSV, pMV, pAdexLcw, retrovirus-derived expression vectors, such as pZIpneo, yeast-derived expression vectors, such as lb
ば pNVll、 SP-QO 枯草菌由来の発現ベクター、 例えば pPL608、 pKTH50、 大腸菌 由来の発現ベクター、 例えば pQE、 pGEAPP、 pGEMEAPP, pMALp2が挙げられる。 本発明のベクターには、 in vivo, in vitroで本発明の蛋白質を製造するのみ ならず、 哺乳動物、 例えばヒトの遺伝子治療に用いるものも含まれる。 Examples include pNVll, an expression vector derived from SP-QO Bacillus subtilis, such as pPL608, pKTH50, and an expression vector derived from Escherichia coli, such as pQE, pGEAPP, pGEMEAPP, and pMALp2. The vectors of the present invention include those used not only for producing the protein of the present invention in vivo and in vitro, but also for gene therapy of mammals, for example, humans.
上述のように構築された本発明の発現べクタ一の宿主への導入方法としては、 公知の方法、 例えばリン酸カルシウム法（Virology ( 1973 ) 52, 456-467)ゃェ レク ト口ポレーシヨン法 （EMBO J. ( 1982 ) 1 , 841-845) 等が用いられる。  As a method for introducing the expression vector of the present invention constructed as described above into a host, known methods, for example, a calcium phosphate method (Virology (1973) 52, 456-467), a selective mouth poration method (EMBO) J. (1982) 1, 841-845) and the like.
本発明において、 蛋白質の製造のために、 任意の産生系を使用することがで きる。蛋白質製造のための産生系としては、 in vitroおよび in vivoの産生系が ある。 in vitroの産生系としては、 真核細胞を使用する産生系や原核細胞を使 用する産生系が挙げられる。  In the present invention, any production system can be used for protein production. Production systems for protein production include in vitro and in vivo production systems. In vitro production systems include production systems using eukaryotic cells and production systems using prokaryotic cells.
真核細胞を使用する場合、 例えば、 動物細胞、 植物細胞、 真菌細胞を用いる 産生系がある。 動物細胞としては、 哺乳類細胞、 例えば CHO (J. Exp. Med. ( 1 995 ) 108, 945) 、 COSヽ ミエローマ、 BHK (baby hamster kidney) 、 HeLaヽ Ve ro、 両生類細胞、 例えばアフリカッメガエル卵母細胞 （Val le, et al .， Natur e ( 1981 ) 291， 358-340) 、 あるいは昆虫細胞、 例えば sf9、 sf21、 Tn5が知られ ている。 CHO細胞としては、 特に DHFR遺伝子を欠損した CH0細胞である dhfr- CH0 (Proc . Natl . Acad. Sci . USA ( 1980 ) 77, 4216-4220) や CHO K-l (Proc. N atl . Acad. Sci . USA ( 1968 ) 60, 1275) を好適に使用することができる。  When eukaryotic cells are used, for example, there are production systems using animal cells, plant cells, and fungal cells. Animal cells include mammalian cells, such as CHO (J. Exp. Med. (1 995) 108, 945), COS ヽ myeloma, BHK (baby hamster kidney), HeLa ヽ Ve ro, amphibian cells, such as African Megafrog Oocytes (Val le, et al., Nature (1981) 291, 358-340) or insect cells such as sf9, sf21, and Tn5 are known. As CHO cells, in particular, DHFR-deficient CH0 cells such as dhfr-CH0 (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO Kl (Proc. Natl. Acad. Sci. USA (1968) 60, 1275) can be suitably used.
植物細胞としては、 ニコチアナ '夕バカム （Nicotiana tabacum) 由来の細胞 が知られており、 これをカルス培養すればよい。 真菌細胞としては、 酵母、 例 えばサッカロミセス （Saccharomyces)属、 例えばサッカロミセス ·セレビシェ (Saccharomyces cerevisiae) 、 糸状困、 ί列； c {まァスぺノレ ノレス属 (Aspergi l lus) 属、 例えばァスペルギルス ·二ガー （Aspergil lus niger) が知られてい る。  As plant cells, cells derived from Nicotiana tabacum are known, and callus culture may be used. Fungal cells include yeast, for example, the genus Saccharomyces, for example, Saccharomyces cerevisiae, filamentous, lineage; c (Aspergillus), for example, Aspergillus niger (Aspergil lus niger) is known.
原核細胞を使用する場合、 細菌細胞を用いる産生系がある。 細菌細胞として は、 大腸菌 （E. col i) 、 枯草菌が知られている。 When prokaryotic cells are used, there are production systems using bacterial cells. As a bacterial cell Escherichia coli (E. coli) and Bacillus subtilis are known.
これらの細胞を目的とする D N Aにより形質転換し、形質転換された細胞を i n vitroで培養することにより蛋白質が得られる。培養は、公知の方法に従い行 う。 例えば、 培養液として、 DMEM、 MEMs RPMI 1640, IMDMを使用することができ る。 その際、 牛胎児血清 （FCS) 等の血清補液を併用することもできるし、 無血 清培養してもよい。 培養時の pHは約 6〜8であるのが好ましい。 培養は通常約 30 〜40°Cで約 15〜200時間行い、 必要に応じて培地の交換、 通気、 攪拌を加える。 一方、 in vivoの産生系としては、動物を使用する産生系や植物を使用する産 生系が挙げられる。 これらの動物又は植物に目的とする D N Aを導入し、 動物 又は植物の体内で蛋白質を産生させ、 回収する。 本発明における 「宿主」 とは、 これらの動物、 植物を包含する。  The protein is obtained by transforming these cells with the desired DNA and culturing the transformed cells in vitro. Culture is performed according to a known method. For example, DMEM, MEMs RPMI 1640, IMDM can be used as a culture solution. At that time, a serum replacement solution such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed. The pH during culturing is preferably about 6-8. Culture is usually performed at about 30 to 40 ° C for about 15 to 200 hours, and the medium is replaced, aerated, and agitated as necessary. On the other hand, examples of in vivo production systems include production systems using animals and production systems using plants. The desired DNA is introduced into these animals or plants, and proteins are produced and recovered in the animals or plants. The “host” in the present invention includes these animals and plants.
動物を使用する場合、 哺乳類動物、 昆虫を用いる産生系がある。 哺乳類動物 としては、 ャギ、 ブ夕、 ヒッジ、 マウス、 ゥシを用いることができる （Vicki Glaser, SPECTRUM Biotechnology Appl ications, 1993)。 また、 哺乳類動物を 用いる場合、 トランスジヱニック動物を用いることができる。  When using animals, there are production systems using mammals and insects. As mammals, goats, bushes, sheep, mice, and mice can be used (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). When a mammal is used, a transgenic animal can be used.
例えば、 目的とする D N Aをャギ ?カゼィンのような乳汁中に固有に産生さ れる蛋白質をコードする遺伝子の途中に挿入して融合遺伝子として調製する。 この D N Aが挿入された融合遺伝子を含む D N A断片をャギの胚へ注入し、 こ の胚を雌のャギへ導入する。 胚を受容したャギから生まれる トランスジェニッ クャギ又はその子孫が産生する乳汁から蛋白質を得る。 トランスジエニックャ ギから産生される蛋白質を含む乳汁量を増加させるために、 適宜ホルモンをト ランスジエニックャギに使用してもよい。 （Ebert, K.M. et al .， Bio/Techno logy ( 1994) 12, 699-702) 。  For example, a desired DNA is inserted into a gene encoding a protein that is uniquely produced in milk, such as goat casein, to prepare a fusion gene. The DNA fragment containing the fusion gene with the inserted DNA is injected into a goat embryo, and the embryo is introduced into a female goat. The protein is obtained from milk produced by the transgenic goat born from the goat that has received the embryo or its progeny. Hormones may be used in the transgenic goat as appropriate to increase the amount of milk containing protein produced from the transgenic goat. (Ebert, K.M. et al., Bio / Technology (1994) 12, 699-702).
また、 昆虫としては、 例えばカイコを用いることができる。 カイコを用いる 場合、 目的とする D N Aを挿入したバキュロウィルスをカイコに感染させ、 こ のカイコの体液より所望の蛋白質を得る （Susumu, M. et al .， Nature ( 1985 ) 1σ In addition, silkworms can be used as insects, for example. When a silkworm is used, the baculovirus into which the target DNA is inserted is infected to the silkworm, and a desired protein is obtained from the body fluid of the silkworm (Susumu, M. et al., Nature (1985)). 1σ
315, 592-594) 。 315, 592-594).
さらに、 植物を使用する場合、 例えばタバコを用いることができる。 タバコ を用いる場合、 目的とする D N Aを植物発現用べクタ一、 例えば ρΜΟΝ 530に挿 入し、 このべクタ一をァグロバクテリゥム ' ヅメファシエンス （Agrobacteriu m tuiefaciens) のようなバクテリアに導入する。 このバクテリアをタバコ、 例 えばニコチアナ .夕バカム （Nicotiana tabacum) に感染させ、 タバコの葉より 所望のポリペプチドを得る （Jul ian, K. -C. Ma et al .， Eur. J. Immunol . ( 1 994) 24, 131-138) 。  Furthermore, when using a plant, for example, tobacco can be used. If tobacco is used, the desired DNA is inserted into a plant expression vector, for example, ρΜΟΝ530, and the vector is introduced into a bacterium such as Agrobacterium m ヅ efaciens. The bacteria are infected with tobacco, for example, Nicotiana tabacum, to obtain the desired polypeptide from tobacco leaves (Julian, K.-C. Ma et al., Eur. J. Immunol. 1 994) 24, 131-138).
上記で得られた本発明の蛋白質は、 細胞内外、 宿主から単離し実質的に純粋 で均一な蛋白質として精製することができる。 蛋白質の分離、 精製は、 通常の 蛋白質の精製で使用されている分離、 精製方法を使用すればよく、 何ら限定さ れるものではない。 例えば、 クロマトグラフィーカラム、 フィルタ一、 限外濾 過、 塩析、 溶媒沈殿、 溶媒抽出、 蒸留、 免疫沈降、 SDS-ポリアクリルアミ ドゲ ル電気泳動、 等電点電気泳動法、 透析、 再結晶等を適宜選択、 組み合わせれば 蛋白質を分離、 精製することができる。  The protein of the present invention obtained as described above can be isolated from a host inside or outside a cell, and purified as a substantially pure and homogeneous protein. The separation and purification of the protein may be performed by the same separation and purification methods used in ordinary protein purification, and is not limited in any way. For example, chromatography columns, filters, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. The proteins can be separated and purified by appropriately selecting and combining.
クロマトグラフィ一としては、 例えばァフィ二ティ一クロマトグラフィー、 イオン交換クロマトグラフィー、 疎水性クロマトグラフィー、 ゲル濾過、 逆相 クロマトグラフィー、 吸着クロマトグラフィー等が挙げられる （Strategies f or Protein Purification and Characterization : A Laboratory Course Manu al . Ed Daniel R. Marshak et al . , Cold Spring Harbor Laboratory Press, 1996) 。 これらのクロマトグラフィーは、 液相クロマトグラフィー、 例えば HP LC、 FPLC等の液相クロマトグラフィーを用いて行うことができる。 本発明は、 これらの精製方法を用い、 高度に精製された蛋白質も包含する。  Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Characterization: A Laboratory Course). Manu al. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory Press, 1996). These chromatographys can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HP LC and FPLC. The present invention also includes highly purified proteins using these purification methods.
なお、 蛋白質を精製前又は精製後に適当なタンパク修飾酵素を作用させるこ とにより、 任意に修飾を加えたり部分的にベプチドを除去することもできる。 タンパク修飾酵素としては、 トリプシン、 キモトリブシン、 リシルエンドぺプ チダ一ゼ、 プロテインキナーゼ、 グルコシダーゼ、 プロテインキナーゼ、 グル コシダ一ゼが用いられる。 The protein can be arbitrarily modified or partially removed by applying an appropriate protein modifying enzyme before or after purification of the protein. Trypsin, chymotrypsin, lysyl endop Tidase, protein kinase, glucosidase, protein kinase, and glucosidase are used.
本発明はまた、 配列番号： 1、 配列番号： 3、 配列番号： 5、 配列番号： 7、 配列番号： 1 9、 及び配列番号： 2 1のいずれかに示されるアミノ酸配列から なる蛋白質の活性中心からなる部分べプチドを含む。 本発明の蛋白質の部分べ プチドとしては、 例えば蛋白質の分子のうち、 疎水性プロッ ト解析から推定さ れる疎水性領域や親水性領域の 1つあるいは複数の領域を含む部分べプチドが 挙げられる。 これらの部分べプチドは 1つの疎水性領域の一部あるいは全部を 含んでいてもよいし、 1つの親水性領域の一部あるいは全部を含んでいてもよ い。 例えば、 本発明の蛋白質の可溶型蛋白質や細胞外領域からなる蛋白質も本 発明に包含される。  The present invention also provides an activity of a protein comprising an amino acid sequence represented by any one of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 19, and SEQ ID NO: 21 Including partial peptides consisting of a center. The partial peptides of the protein of the present invention include, for example, partial peptides containing one or more of a hydrophobic region and a hydrophilic region estimated from a hydrophobic plot analysis in a protein molecule. These partial peptides may include part or all of one hydrophobic region, or may include part or all of one hydrophilic region. For example, a soluble protein of the protein of the present invention and a protein comprising an extracellular region are also included in the present invention.
本発明の蛋白質の部分ペプチドは、 遺伝子工学的手法、 公知のペプチド合成 法、 あるいは本発明の蛋白質を適切なぺプチダーゼで切断することによって製 造することができる。 ペプチド合成法としては、 たとえば固相合成法、 液相合 成法のいずれによっても良い。  The partial peptide of the protein of the present invention can be produced by a genetic engineering technique, a known peptide synthesis method, or by cleaving the protein of the present invention with an appropriate peptidase. As the peptide synthesis method, for example, any of a solid phase synthesis method and a liquid phase synthesis method may be used.
また、 本発明は、 上記本発明の蛋白質をコードする D N Aに関する。 本発明 の蛋白質をコードする c D N Aは、 例えば、 本明細書に記載のプローブを用い ヒト c D N Aライブラリーをスクリーニングして得ることができる。  The present invention also relates to DNA encoding the protein of the present invention. The cDNA encoding the protein of the present invention can be obtained, for example, by screening a human cDNA library using the probe described in the present specification.
得られた c D N A又は c D N A断片をプローブとして、 さらに c D N Aライ ブラリーをスクリーニングすることにより異なる細胞、 組織、 臓器又は種から c D N Aを得ることができる。 c D N Aライブラリ一は、 例えば Sambrook, J. et al . , Molecular Clonings Cold Spring Harbor Laboratory Press ( 1989 ) に記載の方法により調製してもよいし、 市販の D N Aライブラリ一を用いても よい。  By screening the cDNA library using the obtained cDNA or the cDNA fragment as a probe, cDNA can be obtained from different cells, tissues, organs or species. The cDNA library may be prepared, for example, by the method described in Sambrook, J. et al., Molecular Clonings Cold Spring Harbor Laboratory Press (1989), or a commercially available DNA library may be used.
また、 得られた c D N Aの塩基配列を決定することにより、 それがコードす る翻訳領域を決定でき、 本発明の蛋白質のアミノ酸配列を得ることができる。 また、 得られた c DNAをプローブとしてジエノミック DNAライブラリーを スクリーニングすることにより、ジエノミヅク DN Aを単離することができる。 具体的には、 次のようにすればよい。 まず、 本発明の蛋白質を発現する細胞、 組織、 臓器から、 mRNAを単離する。 mRNAの単離は、 公知の方法、 例え ば、 グァニジン超遠心法（Chirgwin, J. M. et al. Biochemistry (1979) 18,Also, by determining the nucleotide sequence of the obtained cDNA, the translation region encoded by the cDNA can be determined, and the amino acid sequence of the protein of the present invention can be obtained. Further, by screening a dienomic DNA library using the obtained cDNA as a probe, dienomic DNA can be isolated. Specifically, the following may be performed. First, mRNA is isolated from cells, tissues, and organs that express the protein of the present invention. The mRNA can be isolated by a known method, for example, guanidine ultracentrifugation (Chirgwin, JM et al. Biochemistry (1979) 18,
5294-5299)、 AGPC法（Chomczynski, P. and Sacchi, N Anal. Biochem. (19 87) 162 156- 159)等により全 RN Aを調製し、 mRNA Purification Kit (Phar macia)等を使用して全 RNAから mRNAを精製する。 また、 QuickPrep mMA5294-5299), the total RNA was prepared by the AGPC method (Chomczynski, P. and Sacchi, N Anal. Biochem. (1987) 162 156-159), etc., and the mRNA was purified using the mRNA Purification Kit (Pharmacia). Purify mRNA from total RNA. Also, QuickPrep mMA
Purification Kit (Pharmacia)を用いることにより mRNAを直接調製する こともできる。 MRNA can also be directly prepared by using Purification Kit (Pharmacia).
得られた mRNAから逆転写酵素を用いて cDNAを合成する。 cDNAの 合成は、 AMV Reverse Transcriptase First-strand c D N A Synthesis Kit (生化学工業） 等を用いて行うこともできる。 また、 本明細書に記載されたプ ローブを用いて、 5 — Ampli FINDER RACE Kit (Clontech製）およびポリメラ一 ゼ連鎖反応 (polymerase chain reaction ； PCR) を用いた 5' - RACE法 (Frohma n M. A. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 8998-9002 ； Belyavsky, A. et al., Nucleic Acids Res. (1989) 17 2919- 2932)にしたが い、 c DNAの合成および増幅を行うことができる。  CDNA is synthesized from the obtained mRNA using reverse transcriptase. cDNA can also be synthesized using AMV Reverse Transcriptase First-Strand DNA Synthesis Kit (Seikagaku). In addition, using the probe described in this specification, the 5′-RACE method (Frohman MA) using the 5 — Ampli FINDER RACE Kit (manufactured by Clontech) and the polymerase chain reaction (PCR) was used. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17 2919-2932) Synthesis and amplification can be performed.
得られた PCR産物から目的とする DN A断片を調製し、ベクター DN Aと連結 する。 さらに、 これより組換えベクターを作製し、 大腸菌等に導入してコロニ 一を選択して所望の組換えベクターを調製する。 目的とする DN Aの塩基配列 を公知の方法、 例えば、 ジデォキシヌクレオチドチェイン夕一ミネーシヨン法 により確認すればよい。  A target DNA fragment is prepared from the obtained PCR product, and ligated with the vector DNA. Further, a recombinant vector is prepared from this, introduced into E. coli, etc., and colonies are selected to prepare a desired recombinant vector. The nucleotide sequence of the target DNA may be confirmed by a known method, for example, the dideoxynucleotide chain-one-minute method.
また、本発明の DNAは、発現に使用する宿主のコドン使用頻度を考慮して、 より発現効率の高い配列を設計することができる （Grantham, R. et al., Nuc elic Acids Research (1981) 9 D43-p74)。 また、 本発明の DNAを市販のキ ッ トゃ公知の方法によって改変することができる。 例えば、 制限酵素による消 化、 合成オリゴヌクレオチドや適当な DN Aフラグメントの挿入、 リンカ一の 付加、 開始コ ドン （ATG) 及び/又は終始コ ドン （ATT、 TGA又は TAG) の挿入等 が挙げられる。 In addition, the DNA of the present invention can be designed to have a higher expression efficiency in consideration of the codon usage of the host used for expression (Grantham, R. et al., Nucelic Acids Research (1981)). 9 D43-p74). In addition, the DNA of the present invention is ゃ can be modified by a known method. Examples include digestion with a restriction enzyme, insertion of a synthetic oligonucleotide or an appropriate DNA fragment, addition of a linker, insertion of an initiation codon (ATG) and / or a termination codon (ATT, TGA or TAG), and the like. .
本発明の DN Aは、具体的には配列番号： 2の塩基配列において 441位の塩基 Aから 1523位の塩基 Cからなる DNA、配列番号： 4の塩基配列において 441位の Aから 872位の塩基 Aからなる DNA、 配列番号： 6の塩基配列において 659位の 塩基 Aから 1368位の塩基 Cからなる D N A及び配列番号： 8の塩基配列において 4 41位の塩基 Aから 2054位の塩基 Cからなる DNA、 配列番号： 2 0の塩基配列に おいて 439位の塩基 Aから 870位の塩基 Aからなる D NA、 及び配列番号： 22の 塩基配列において 439位の塩基 Aから 2052位の塩基 Cからなる DN Aを包含する。 本発明の DN Aはまた、 配列番号： 2、 配列番号： 4、 配列番号： 6、 配列 番号： 8、 配列番号： 2 0、 及び配列番号： 22から 27のいずれかに示す塩 基配列からなる DNAとストリンジェントな条件下でハイブリダイズする D N Aであり、且つ上記 NR8蛋白質と機能的に同等な蛋白質をコ一ドする DNAを含 む。  Specifically, the DNA of the present invention is a DNA consisting of the base A at position 441 to the base C at position 1523 in the base sequence of SEQ ID NO: 2, the base sequence of SEQ ID NO: 4 DNA consisting of base A, DNA consisting of base A at position 659 in the base sequence of SEQ ID NO: 6 to base C of position 1368 and base C of base 41 of position 41 to base 2054 in base sequence SEQ ID NO: 8 DNA consisting of base A at position 439 in the base sequence of SEQ ID NO: 20 to base A at position 870 in the base sequence of SEQ ID NO: 20, and base C of base A at position 439 to base 2052 in the base sequence of SEQ ID NO: 22 A DNA consisting of: The DNA of the present invention also includes a nucleotide sequence represented by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20 and SEQ ID NO: 22 to 27. DNA that hybridizes with other DNAs under stringent conditions and that encodes a protein functionally equivalent to the NR8 protein.
ストリンジェン卜な条件としては、 当業者であれば適宜選択することができ るが、 例えば低ス トリンジェン卜な条件が挙げられる。 低ス ト リンジェン卜の 条件とは、 例えば 42°C、 2xSSC、 0.1%SDSが挙げられ、 好ましくは 50°C、 2xS SC、 0.1%SDSである。 またより好ましくは、 高ストリンジェントな条件が挙げ られる。 高ストリンジェン卜な条件とは、 例えば 65° 2XSSC及び 0.1%SDSが 挙げられる。 これらの条件において、 温度を上げる程に高い相同性を有する D N Aを得ることができる。 上記のハイブリダィズする D N Aは好ましくは天然 由来の DNA、 例えば c DNA又は染色体 DNAである。  Stringent conditions can be appropriately selected by those skilled in the art, and examples include low stringent conditions. The conditions of low stringency include, for example, 42 ° C., 2 × SSC, 0.1% SDS, and preferably 50 ° C., 2 × SSC, 0.1% SDS. More preferably, high stringency conditions are used. Highly stringent conditions include, for example, 65 ° 2XSSC and 0.1% SDS. Under these conditions, DNA with higher homology can be obtained as the temperature is increased. The hybridizing DNA is preferably a naturally occurring DNA, such as cDNA or chromosomal DNA.
実施例に示すごとく、 本発明の蛋白質をコ一ドする c DNAとハイプリダイ ズする遺伝子の mRN Aは、 ヒ卜の種々の組織にも分布していることが判明し ムム As shown in the Examples, it was found that mRNA of a gene that hybridizes with cDNA encoding the protein of the present invention was also distributed in various tissues of humans. Mum
た。 従って、 上記の天然由来の DN Aは、 例えば実施例において本発明の蛋白 質をコードする c DNAとハイプリダイズする mRNAが検出される組織由来 の cDNAゃジエノミック DNAであってもよい。 また、 本発明の蛋白質をコ —ドする DNAは、 cDNAゃジエノミック DNAの他、 合成 DNAであって もよい。 Was. Therefore, the above-mentioned naturally-derived DNA may be, for example, a cDNA dienomic DNA derived from a tissue in which mRNA that hybridizes with the cDNA encoding the protein of the present invention in Examples is detected. Further, the DNA encoding the protein of the present invention may be a synthetic DNA in addition to the cDNA dienomic DNA.
本発明の蛋白質は、 これに結合する化合物のスクリーニングに有用である。 すなわち、 本発明の蛋白質と、 該蛋白質に結合する化合物を含むと予想される 被験試料とを接触せしめ、 そして本発明の蛋白質に結合する活性を有する化合 物を選択する、 ことからなる本発明の蛋白質に結合する化合物をスクリーニン グする方法において使用される。  The protein of the present invention is useful for screening for a compound that binds to the protein. That is, the protein of the present invention is brought into contact with a test sample expected to contain a compound binding to the protein, and a compound having an activity of binding to the protein of the present invention is selected. Used in methods to screen for compounds that bind to proteins.
本発明の蛋白質に結合する活性を有する化合物をスクリーニングする方法と しては、 当業者に通常用いられる多くの方法を使用することができる。 本発明 のスクリーニングに用いられる本発明の蛋白質は組換え型、 天然型又は部分べ プチドのいずれであってもよい。 また、 本発明の蛋白質に結合する活性を有す る化合物としては、 結合活性を有する蛋白質であってもよく、 あるいは結合活 性を有する化学合成された化合物であってもよい。  As a method for screening for a compound having an activity of binding to the protein of the present invention, many methods commonly used by those skilled in the art can be used. The protein of the present invention used in the screening of the present invention may be any of a recombinant type, a natural type and a partial peptide. Further, the compound having an activity of binding to the protein of the present invention may be a protein having a binding activity, or may be a chemically synthesized compound having a binding activity.
また、 本発明のスクリーニング方法で使用される被験試料としては、 例えば ペプチド、 精製若しくは粗精製蛋白質、 非ペプチド性化合物、 合成化合物、 微 生物発酵生産物、 海洋生物抽出液、 植物抽出液、 細胞抽出液、 動物組織抽出液 が挙げられる。 これらの被験試料は新規な化合物であってもよいし、 公知の化 合物であってもよい。  The test sample used in the screening method of the present invention includes, for example, peptides, purified or partially purified proteins, non-peptide compounds, synthetic compounds, fermentation products of microorganisms, marine organism extracts, plant extracts, and cell extracts. Liquid and animal tissue extract. These test samples may be novel compounds or known compounds.
本発明の蛋白質と結合する蛋白質のスクリーニングは、 例えば、 ウェストウ エスタンブロッテイング法（Skolnik， E. Y. et al.， Cell ( 1991 ) 65， 83-90) を用いて行うことができる。 本発明の蛋白質と結合する蛋白質を発現している と予想される細胞、 組織、 臓器より cDNAを単離し、 これをファージベクタ 一、 例えば入 gtll、 ZAPII等へ導入して c DNAライブラリーを作製し、 これを 培地を引いたプレート上で発現させ、フィル夕一に発現させた蛋白質を固定し、 標識して精製した本発明の蛋白質と上記フィル夕一とを反応させ、 本発明の蛋 白質と結合した蛋白質を発現するプラークを標識により検出すればよい。 本発 明の蛋白質を標識する方法としては、 ピオチンとアビジンの結合性を利用する 方法、 本発明の蛋白質又は本発明の蛋白質に融合したぺプチド又はポリべプチ ドに特異的に結合する抗体を利用する方法、 ラジオァイソ トープを利用する方 法又は蛍光を利用する方法等が挙げられる。 Screening of a protein that binds to the protein of the present invention can be performed, for example, using the Westwestern blotting method (Skolnik, EY et al., Cell (1991) 65, 83-90). A cDNA is isolated from a cell, tissue, or organ that is expected to express a protein that binds to the protein of the present invention, and introduced into a phage vector, for example, gtll, ZAPII, etc., to produce a cDNA library. And this A protein bound to the protein of the present invention, which is expressed on a plate from which a medium has been drawn, and the protein of the present invention, which has been expressed and fixed on a plate, is allowed to react with the labeled and purified protein of the present invention, and the above-mentioned filter, and binds to the protein of the present invention. May be detected by labeling the plaques expressing. Examples of the method for labeling the protein of the present invention include a method using the binding property of biotin and avidin, and an antibody that specifically binds to the protein of the present invention or a peptide or polypeptide fused to the protein of the present invention. A method using a radioisotope, a method using fluorescence, and the like.
また、 本発明の蛋白質と特異的に結合するリガンドのスクリーニングは、 本 発明の蛋白質の細胞外ドメインと既知のシグナル伝達能を有するへモポェチン 受容体蛋白質の細胞膜貫通ドメインを含む細胞内ドメインとを連結せしめて作 製したキメラ受容体を、 適当な細胞株、 好ましくは適当な増殖因子の存在下で のみ生存および増殖可能な細胞株 （増殖因子依存性細胞株） の細胞表面に発現 せしめた後、 該細胞株を種々の増殖因子、 サイ ト力イン、 または造血因子等を 含むことが期待される材料を添加して培養することにより実施可能である。 こ の方法は、 被検材料中に本発明蛋白質の細胞外ドメインと特異的に結合するリ ガンドが存在する場合にのみ、 上記増殖因子依存性細胞株が生存および増殖が 可能であることを利用している。既知のへモポェチン受容体としては、例えば、 トロンボポェチン受容体、 エリスロポエチン受容体、 G- CSF受容体、 gP130等が 挙げられるが、 本発明のスクリーニング系に用いるキメラ受容体のパートナー は、 これら既知のへモポェチン受容体に限定されるものではなく、 細胞質ドメ インにシグナル伝達活性に必要な構造を備えているものであれば何を用いても 構わない。 増殖因子依存性細胞株としては、 例えば、 BaF3や FDC- P1を初めとし た IL3依存性細胞株を利用することが可能である。 Screening for a ligand that specifically binds to the protein of the present invention comprises linking the extracellular domain of the protein of the present invention with the intracellular domain of the hemopoietin receptor protein having a known signal transduction ability, including the transmembrane domain. After expressing the chimeric receptor produced at least on the cell surface of a suitable cell line, preferably a cell line that can survive and grow only in the presence of a suitable growth factor (growth factor-dependent cell line), This can be carried out by culturing the cell line with the addition of a material expected to contain various growth factors, cytodynamic factors, hematopoietic factors and the like. This method utilizes the fact that the growth factor-dependent cell line can survive and proliferate only when the test material contains a ligand that specifically binds to the extracellular domain of the protein of the present invention. are doing. The Mopoechin receptor to known for, for example, Toronbopoechin receptor, erythropoietin receptor, G-CSF receptor, but g P 130, and the like, partners of the chimeric receptor used in the screening system of the present invention, these known The present invention is not limited to the hemopoietin receptor, and any cytoplasmic domain having a structure necessary for signal transduction activity may be used. As a growth factor-dependent cell line, for example, an IL3-dependent cell line such as BaF3 or FDC-P1 can be used.
本発明の蛋白質と特異的に結合するリガンドとしては、 希ではあるが可溶性 蛋白質ではなく細胞膜結合型蛋白質である可能性も想定される。 この様な場合 にはむしろ本発明の蛋白質の細胞外ドメインのみを含む蛋白質あるいは当該細 胞外ドメインに他の可溶性蛋白質の部分配列を付加した融合蛋白質を標識後、 リガンドを発現していることが期待される細胞との結合を測定することにより スクリーニングすることが可能である。 本発明の蛋白質の細胞外ドメインのみ を含む蛋白質としては、 例えば、 細胞膜貫通ドメインの N端側に終止コドンを 挿入することにより人為的に作成した可溶性受容体蛋白質、あるいは NR8 3の可 溶型蛋白質が利用可能である。 一方、 本発明の蛋白質の細胞外ドメインに他の 可溶性蛋白質の部分配列を付加した融合蛋白質としては、 例えば、 免疫グロブ リンの Fc部位や FLAGぺプチド等を細胞外ドメインの C端に付加して調製した蛋 白質が利用可能である。 これらの可溶性標識蛋白質は上述したウェストウエス タン法における検出にも利用可能である。 As a ligand that specifically binds to the protein of the present invention, it is conceivable that a rare but soluble protein may be a cell membrane-bound protein. In such a case, rather, the protein containing only the extracellular domain of the protein of the present invention or the protein containing Screening can be performed by labeling a fusion protein obtained by adding a partial sequence of another soluble protein to the extracellular domain, and then measuring the binding to a cell expected to express the ligand. Examples of the protein containing only the extracellular domain of the protein of the present invention include a soluble receptor protein artificially prepared by inserting a stop codon at the N-terminal side of the transmembrane domain, or a soluble NR83 protein Is available. On the other hand, as a fusion protein in which a partial sequence of another soluble protein is added to the extracellular domain of the protein of the present invention, for example, an Fc site of immunoglobulin, a FLAG peptide or the like is added to the C-terminal of the extracellular domain. The prepared protein can be used. These soluble labeled proteins can also be used for detection in the above-mentioned West Western method.
本発明の蛋白質に結合する蛋白質のスクリーニングは、 two-ハイブリツ ドシ ステム (Fields, S.， and Sternglanz, R. , Trends. Genet. ( 1994) 10, 286- 292) を用いて行うこともできる。  Screening for proteins that bind to the protein of the present invention can also be performed using a two-hybrid system (Fields, S., and Sternglanz, R., Trends. Genet. (1994) 10, 286-292). .
two-ハイブリツ ドシステムにおいては、 本発明の蛋白質とヘテロダイマーか らなる転写調節因子の一方のサブュニッ トとの融合蛋白質をコードする D N A を含む発現ベクター及び被験試料として所望の c D N Aとへテロダイマ一から なる転写調節因子のもう一方のサブュニットをコードする D N Aを連結してな る D N Aを含む発現べクタ一を細胞に導入して発現させる。 本発明の蛋白質に c D N Aがコードする蛋白質が結合して該転写調節因子がヘテロダイマーを形 成した場合、 あらかじめ細胞内に構築したレポーター遺伝子が発現する。 従つ て、 本発明の蛋白質に結合する蛋白質を、 レポーター遺伝子の発現量を検出又 は測定することにより選択することができる。  In the two-hybrid system, an expression vector containing a DNA encoding a fusion protein of the protein of the present invention and one of the subunits of a transcription factor comprising a heterodimer, and a desired cDNA as a test sample and a heterodimer. An expression vector containing DNA obtained by ligating the DNA encoding the other subunit of the transcriptional regulatory factor is introduced into cells and expressed. When the protein encoded by the cDNA binds to the protein of the present invention to form a heterodimer, the reporter gene previously constructed in the cell is expressed. Therefore, a protein that binds to the protein of the present invention can be selected by detecting or measuring the expression level of a reporter gene.
具体的には、 次のようにすればよい。 すなわち、 本発明の蛋白質をコードす る D N Aと LexAの D N A結合ドメインをコードする遺伝子とをフレームが一致 するように連結し、 発現べクタ一を作製する。 次に、 所望の c D N Aと GAL4転 写活性化ドメインをコードする遺伝子とを連結せしめることにより発現べク夕 —を作製する。 Specifically, the following may be performed. That is, a DNA encoding the protein of the present invention and a gene encoding the DNA binding domain of LexA are ligated in such a manner that the frames match, and an expression vector is prepared. Next, the expression vector is ligated by linking the desired cDNA to the gene encoding the GAL4 transcription activation domain. -Is prepared.
LexA結合モチーフが存在するプロモ一夕一により転写が調節される HIS3遺伝 子を組み込んだ細胞を上記の two-ハイプリッ ドシステム発現プラスミ ドを用い て形質転換した後、 ヒスチジン不含合成培地上でィンキュベ一卜するとタンパ クの相互作用が認められたときのみ細胞の生育が観察される。 このように、 形 質転換体の生育程度によりレポ一夕一遺伝子の発現量の増加を調べることがで ぎる。  Cells transformed with the HIS3 gene, whose transcription is regulated by a promoter that has a LexA-binding motif, are transformed using the two-hybrid system expression plasmid described above, and then transformed into a histidine-free synthetic medium. In short, cell growth is observed only when protein interaction is observed. Thus, it is possible to examine the increase in the expression level of the repo overnight gene depending on the degree of growth of the transformants.
レポ—夕—遺伝子としては、 HIS3遺伝子の他、 例えば、 ルシフヱラーゼ遺伝 子、 PAI-1 (Plasminogen activator inhibitor typel) 遺伝子、 ADE2遺伝子、 LacZ遺伝子、 CDC25H遺伝子等を用いることができる。  As the repo-gene, in addition to the HIS3 gene, for example, luciferase gene, PAI-1 (Plasminogen activator inhibitor typel) gene, ADE2 gene, LacZ gene, CDC25H gene and the like can be used.
two-hybrid systemは、通常用いられている方法により構築してもよいし、巿 販のキヅ トを用いてもよい。 市販の two-ハイブリッ ドシステムのキヅトとして は、 ATCHMARKER Two-Hybrid System, Mammal ian MATCHMARKER Two-Hybrid As say Kit (いずれも CLONTECH製） 、 HybriZAP Two-Hybrid Vector System (Stra tagene製) 、 CytoTrap two-hybrid system ( Stratagene製) が挙げられる。 本発明の蛋白質に結合する蛋白質のスクリーニングは、 ァフィ二テイクロマ トグラフィ一を用いて行うこともできる。 すなわち、 本発明の蛋白質をァフィ 二ティ一カラムの担体に固定し、 ここに本発明の蛋白質と結合する蛋白質を発 現していることが予想される被験試料を適用する。 この場合の被験試料として は、 細胞の培養上清、 細胞抽出物、 細胞溶解物等が挙げられる。 被験試料を適 用した後、 カラムを洗浄し、 本発明の蛋白質に結合した蛋白質を得ることがで きる。  The two-hybrid system may be constructed by a commonly used method, or may use a commercially available kit. Commercially available two-hybrid system kits include ATCHMARKER Two-Hybrid System, Mammalian MATCHMARKER Two-Hybrid As say Kit (all manufactured by CLONTECH), HybriZAP Two-Hybrid Vector System (manufactured by Stra tagene), CytoTrap two-hybrid system (manufactured by Stratagene). Screening for a protein that binds to the protein of the present invention can also be carried out using affinity chromatography. That is, the protein of the present invention is immobilized on a carrier of an affinity column, and a test sample which is expected to express a protein that binds to the protein of the present invention is applied here. Test samples in this case include cell culture supernatants, cell extracts, cell lysates, and the like. After applying the test sample, the column is washed to obtain the protein bound to the protein of the present invention.
本発明のスクリーニング方法によって単離される化合物は、 本発明の蛋白質 の活性を促進又は阻害するための薬剤の候補となる。 本発明のスクリーニング 方法を用いて得られる、 本発明の蛋白質に結合する活性を有する化合物の構造 の一部を、 付加、 欠失及び/又は置換により変換される化合物も、 本発明のス クリーニング方法を用いて得られる化合物に含まれる。 The compound isolated by the screening method of the present invention is a candidate for a drug for promoting or inhibiting the activity of the protein of the present invention. Compounds obtained by using the screening method of the present invention and having a part of the structure of the compound having the activity of binding to the protein of the present invention, which are converted by addition, deletion and / or substitution, also include the compounds of the present invention. Included in compounds obtained using cleaning methods.
本発明のスクリーニング方法を用いて得られる化合物をヒトゃ他の哺乳動物、 例えばマウス、 ラッ ト、 モルモヅ ト、 ゥサギ、 ニヮ トリ、 ネコ、 ィヌ、 ヒヅジ、 ブ夕、 ゥシ、 サル、 マントヒヒ、 チンパンジーの医薬として使用する場合、 常 用される手段に従って実施することができる。  Compounds obtained by the screening method of the present invention can be used for humans and other mammals, such as mice, rats, guinea pigs, egrets, chicks, cats, dogs, sheep, bush, sea lions, monkeys, and baboons. When used as a chimpanzee medicament, it can be carried out according to commonly used means.
例えば、 必要に応じて糖衣を施した錠剤、 カプセル剤、 エリキシル剤、 マイ クロカプセル剤として経口的に、 あるいは水もしくはそれ以外の薬学的に許容 し得る液との無菌性溶液、又は懸濁液剤の注射剤の形で非経口的に使用できる。 例えば本発明の蛋白質と結合活性を有する化合物を生理学的に認められる担体、 香味剤、 賦形剤、 べヒクル、 防腐剤、 安定剤、 結合剤とともに一般に認められ た製薬実施に要求される単位用量形態で混和することによって製造することが できる。 これら製剤における有効成分量は指示された範囲の適当な容量が得ら れるようにするものである。  For example, tablets, capsules, elixirs, and microcapsules, which are sugar-coated as required, orally, or aseptic solution or suspension in water or other pharmaceutically acceptable liquids Can be used parenterally in the form of injections. For example, a unit dose required for pharmaceutical practice generally recognized as a physiologically acceptable carrier, flavoring agent, excipient, vehicle, preservative, stabilizer, and binder together with a compound having binding activity to the protein of the present invention. It can be produced by mixing in the form. The amount of the active ingredient in these preparations is such that an appropriate dose in the specified range can be obtained.
錠剤、 力プセル剤に混和することができる添加剤としては、例えばゼラチン、 コーンスターチ、 トラガントガム、 アラビアゴムのような結合剤、 結晶性セル ロースのような賦形剤、 コーンスターチ、 ゼラチン、 アルギン酸のような膨化 剤、 ステアリン酸マグネシウムのような潤滑剤、 ショ糖、 乳糖又はサッカリン のような甘味剤、 ペパーミント、 ァカモノ油又はチェリーのような香味剤が用 いられる。 調剤単位形態がカプセルである場合には、 上記の材料にさらに油脂 のような液状担体を含有することができる。 注射のための無菌組成物は注射用 蒸留水のようなべヒクルを用いて通常の製剤実施に従って処方することができ る。  Excipients that can be incorporated into tablets and capsules include, for example, binders such as gelatin, corn starch, tragacanth gum, acacia, excipients such as crystalline cellulose, corn starch, gelatin, alginic acid and the like. Swelling agents, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, and flavoring agents such as peppermint, cocoa oil or cherry are used. When the unit dosage form is a capsule, the above materials may further contain a liquid carrier such as oil and fat. Sterile compositions for injection can be formulated according to normal pharmaceutical practice using a vehicle such as distilled water for injection.
注射用の水溶液としては、 例えば生理食塩水、 ブドウ糖やその他の補助薬を 含む等張液、 例えば D-ソルビトール、 D-マンノース、 D-マンニトール、 塩化ナ トリウムが挙げられ、 適当な溶解補助剤、 例えばアルコール、 具体的にはエタ ノール、 ポリアルコール、 例えばプロピレングリコール、 ポリエチレングリコ —ル、 非イオン性界面活性剤、 例えばポリソルベート 80 ( TM) 、 HCO-50と併用 してもよい。 Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and suitable solubilizing agents, For example, alcohols, specifically ethanol, polyalcohols, such as propylene glycol, polyethylene glyco Or a nonionic surfactant such as polysorbate 80 (TM) or HCO-50.
油性液としてはゴマ油、 大豆油があげられ、 溶解補助剤として安息香酸ベン ジル、 ベンジルアルコールと併用してもよい。 また、 緩衝剤、 例えばリン酸塩 緩衝液、 酢酸ナトリウム緩衝液、 無痛化剤、 例えば、 塩酸プロ力イン、 安定剤、 例えばべンジルアルコール、 フヱノール、 酸化防止剤と配合してもよい。 調製 された注射液は通常、 適当なアンプルに充填させる。  Oily liquids include sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. It may also be combined with a buffer, for example, a phosphate buffer, a sodium acetate buffer, a soothing agent, for example, proforce hydrochloride, a stabilizer, for example, benzyl alcohol, phenol, or an antioxidant. The prepared injection solution is usually filled into an appropriate ampoule.
本発明の蛋白質と結合活性を有する化合物の投与量は、 症状により差異はあ るが、 経口投与の場合、 一般的に成人 （体重 60kgとして） においては、 1日あた り約 0. 1から 100mg、 好ましくは約 1 . 0から 50mg、 より好ましくは約 1. 0から 20mg である。  The dose of the compound having the binding activity to the protein of the present invention varies depending on the symptom. However, in the case of oral administration, generally, for an adult (assuming a body weight of 60 kg), the dose is from about 0.1 per day. 100 mg, preferably about 1.0 to 50 mg, more preferably about 1.0 to 20 mg.
非経口的に投与する場合は、 その 1回投与量は投与対象、 対象臓器、 症状、 投与方法によっても異なるが、 例えば注射剤の形では通常成人 （体重 60kgとし て） においては、 1日あたり約 0. 01から 30mg、 好ましくは約 0. 1から 20mg、 より 好ましくは約 0. 1から lOmg程度を静脈注射により投与するのが好都合である。他 の動物の場合も、 体重 60kg当たりに換算した量を投与することができる。  In the case of parenteral administration, the single dose varies depending on the subject of administration, target organ, symptoms, and administration method. For example, in the case of parenteral injections, usually for adults (with a body weight of 60 kg) per day, It is convenient to administer about 0.01 to 30 mg, preferably about 0.1 to 20 mg, more preferably about 0.1 to 10 mg by intravenous injection. In the case of other animals, the dose can be administered in terms of the weight per 60 kg.
本発明の抗体は、 公知の手段を用いてモノクローナル抗体又はポリクロ一ナ ル抗体として得ることができる。  The antibody of the present invention can be obtained as a monoclonal antibody or a polyclonal antibody using known means.
本発明の蛋白質に対して特異的に結合する抗体は、 蛋白質を感作抗原として 使用して、 これを通常の免疫方法にしたがって免疫し、 得られる免疫細胞を通 常の細胞融合法によって公知の親細胞と融合させ、 通常のスクリ一ニング法に より、 抗体産生細胞をスクリーニングすることによって作製できる。  An antibody that specifically binds to the protein of the present invention can be obtained by immunizing the protein using the protein as a sensitizing antigen in accordance with a usual immunization method and obtaining the obtained immune cells by a conventional cell fusion method. It can be produced by fusing with a parent cell and screening antibody-producing cells by a usual screening method.
具体的には、 本発明の蛋白質に対して特異的に結合するモノクローナル抗体 又はポリクローナル抗体を作製するには次のようにすればよい。  Specifically, a monoclonal or polyclonal antibody that specifically binds to the protein of the present invention may be prepared as follows.
例えば、 抗体取得の感作抗原として使用される本発明の蛋白質は、 その由来 となる動物種に制限されないが哺乳動物、 例えばヒト、 マウス又はラッ ト由来 の蛋白質が好ましく、特にヒト由来の蛋白質が好ましい。ヒト由来の蛋白質は、 本明細書に開示される遺伝子配列又はアミノ酸配列を用いて得ることができる。 本発明において、 感作抗原として使用される蛋白質は、 本明細書に記載され た全ての蛋白質の生物学的活性を有する蛋白質を使用できる。 また、 蛋白質の 部分ペプチドも用いることができる。 蛋白質の部分ペプチドとしては、 例えば 蛋白質のアミノ基 （N) 末端断片やカルボキシ （C) 末端断片が挙げられる。 本 明細書で述べる 「抗体」 とは蛋白質の全長又は断片に特異的に反応する抗体を 意味する。 For example, the protein of the present invention used as a sensitizing antigen for obtaining an antibody is not limited to an animal species from which the protein is derived, but may be derived from a mammal such as a human, a mouse or a rat. Are preferred, and human-derived proteins are particularly preferred. A human-derived protein can be obtained using the gene sequence or amino acid sequence disclosed herein. In the present invention, as the protein used as the sensitizing antigen, a protein having the biological activity of any of the proteins described in the present specification can be used. Further, a partial peptide of a protein can also be used. Examples of the partial peptide of the protein include an amino group (N) terminal fragment and a carboxy (C) terminal fragment of the protein. As used herein, “antibody” refers to an antibody that specifically reacts with the full length or fragment of a protein.
本発明の蛋白質又はその断片をコードする遺伝子を公知の発現べクタ一系に 挿入して本明細書で述べた宿主細胞を形質転換させた後、 その宿主細胞内外又 は、 宿主から目的の蛋白質又はその断片を公知の方法で得、 この蛋白質を感作 抗原として用いればよい。 また、 蛋白質を発現する細胞又はその溶解物あるい は化学的に合成した本発明の蛋白質を感作抗原として使用してもよい。  After the gene encoding the protein of the present invention or a fragment thereof is inserted into a known expression vector system and the host cell described in this specification is transformed, the desired protein can be transferred inside or outside the host cell or from the host. Alternatively, a fragment thereof may be obtained by a known method, and this protein may be used as a sensitizing antigen. Alternatively, a cell expressing the protein, a lysate thereof, or a chemically synthesized protein of the present invention may be used as the sensitizing antigen.
感作抗原で免疫される哺乳動物としては、 特に限定されるものではないが、 細胞融合に使用する親細胞との適合性を考慮して選択するのが好ましく、 一般 的にはげつ歯目、 ゥサギ目、 霊長目の動物が使用される。  The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion.ゥ Egrets and primates are used.
げっ歯目の動物としては、 例えば、 マウス、 ラッ ト、 ハムスター等が使用さ れる。 ゥサギ目の動物としては、 例えば、 ゥサギが使用される。 霊長目の動物 としては、 例えばサルが使用される。 サルとしては、 狭鼻下目のサル （旧世界 ザル） 、 例えば、 力二クイザル、 ァカゲザル、 マントヒヒ、 チンパンジー等が 使用される。  As rodent animals, for example, mice, rats, hamsters and the like are used.動物 As an heronoid animal, for example, a heron is used. For example, monkeys are used as primates. As the monkeys, monkeys of the lower nose (old world monkeys), for example, cynomolgus monkeys, macaques, baboons, and chimpanzees are used.
感作抗原を動物に免疫するには、公知の方法にしたがって行われる。例えば、 一般的方法として、 感作抗原を哺乳動物の腹腔内又は、 皮下に注射することに より行われる。 具体的には、 感作抗原を PBS (Phosphate-Buffered Saline ) や 生理食塩水等で適当量に希釈、懸濁したものを所望により通常のアジュバント、 例えば、 フロイント完全アジュバントを適量混合し、 乳化後、 哺乳動物に投与 し、以降フロイント不完全アジュバン卜に適量混合した感作抗原を、 4〜21日毎 に数回投与するのが好ましい。 また、 感作抗原免疫時に適当な担体を使用する ことができる。 このように免疫し、 血清中に所望の抗体レベルが上昇するのを 常法により確認する。 Immunization of an animal with a sensitizing antigen is performed according to a known method. For example, as a general method, a sensitizing antigen is injected intraperitoneally or subcutaneously into a mammal. Specifically, the sensitizing antigen is diluted and suspended in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and then mixed with an ordinary adjuvant, for example, Freund's complete adjuvant, if desired, and then emulsified. Administered to mammals Thereafter, it is preferable to administer the sensitizing antigen mixed with an appropriate amount of Freund's incomplete adjuvant several times every 4 to 21 days. In addition, a suitable carrier can be used at the time of immunization with the sensitizing antigen. Immunization is performed in this manner, and an increase in the level of the desired antibody in the serum is confirmed by a conventional method.
ここで、 本発明の蛋白質に対するポリクローナル抗体を得るには、 血清中の 所望の抗体レベルが上昇したことを確認した後、 抗原を感作した哺乳動物の血 液を取り出す。 この血液から公知の方法により血清を分離する。 ポリクローナ ル抗体としてポリクローナル抗体を含む血清を使用してもよいし、 必要に応じ この血清からポリクロ一ナル抗体を含む画分をさらに単離してもよい。  Here, in order to obtain a polyclonal antibody against the protein of the present invention, after confirming that the level of the desired antibody in the serum has increased, the blood of a mammal sensitized with the antigen is taken out. The serum is separated from the blood by a known method. A serum containing the polyclonal antibody may be used as the polyclonal antibody, and if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum.
モノクローナル抗体を得るには、 上記抗原を感作した哺乳動物の血清中に所 望の抗体レベルが上昇するのを確認した後に、 哺乳動物から免疫細胞を取り出 し、 細胞融合に付せばよい。 この際、 細胞融合に使用される好ましい免疫細胞 として、 特に脾細胞が挙げられる。 前記免疫細胞と融合される他方の親細胞と しては、 好ましくは哺乳動物のミエローマ細胞、 より好ましくは、 薬剤による 融合細胞選別のための特性を獲得したミエローマ細胞が挙げられる。  To obtain a monoclonal antibody, after confirming that the desired antibody level is increased in the serum of the mammal sensitized with the antigen, the immune cells may be removed from the mammal and subjected to cell fusion. . At this time, spleen cells are particularly preferable as the immune cells used for cell fusion. The other parent cell to be fused with the immune cell is preferably a mammalian myeloma cell, and more preferably a myeloma cell that has acquired the properties for selecting fused cells by a drug.
前記免疫細胞とミエローマ細胞の細胞融合は基本的には公知の方法、例えば、 ミルスティンらの方法（Galfre, G. and Mi lstein, C .， Methods Enzymol . ( 19 81 ) 73, 3-46 ) 等に準じて行うことができる。  The cell fusion of the immune cells and myeloma cells is basically performed by a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol. (1981) 73, 3-46) and the like. It can be performed according to.
細胞融合により得られたハイプリ ドーマは、 通常の選択培養液、 例えば HAT 培養液 （ヒポキサンチン、 アミノプテリンおよびチミジンを含む培養液） で培 養することにより選択される。 当該 H A T培養液での培養は、 目的とするハイ プリ ドーマ以外の細胞 （非融合細胞） が死滅するのに十分な時間、 通常数日〜 数週間継続する。 ついで、 通常の限界希釈法を実施し、 目的とする抗体を産生 するハイプリ ドーマのスクリーニングおよびクローニングが行われる。  The hybridoma obtained by cell fusion is selected by culturing in a normal selective culture medium, for example, a HAT culture medium (a culture medium containing hypoxanthine, aminopterin and thymidine). The cultivation in the HAT culture solution is continued for a period of time sufficient to kill cells (non-fused cells) other than the desired hybridoma, usually several days to several weeks. Next, a conventional limiting dilution method is performed, and screening and cloning of hybridomas producing the desired antibody are performed.
また、 ヒ ト以外の動物に抗原を免疫して上記ハイプリ ドーマを得る他に、 ヒ トリンパ球、 例えば EBウィルスに感染したヒトリンパ球を in vitroで蛋白質、 蛋白質発現細胞又はその溶解物で感作し、 感作リンパ球をヒト由来の永久分裂 能を有するミエローマ細胞、 例えば U266と融合させ、 蛋白質への結合活性を有 する所望のヒ卜抗体を産生するハイプリ ドーマを得ることもできる （特開昭 63 -17688) 。 In addition, in addition to obtaining the above-mentioned hybridoma by immunizing animals other than humans with the antigen, human lymphocytes, for example, human lymphocytes infected with the EB virus, Sensitize with protein-expressing cells or a lysate thereof, and fuse the sensitized lymphocytes with human-derived myeloma cells having permanent division ability, such as U266, to produce a desired human antibody having protein binding activity Hypri-doma can also be obtained (JP-A-63-17688).
さらに、 ヒト抗体遺伝子のレバ一トリーを有するトランスジエニック動物に 抗原となる蛋白質、 蛋白質発現細胞又はその溶解物を免疫して抗体産生細胞を 取得し、 これをミエローマ細胞と融合させたハイプリ ドーマを用いて蛋白質に 対するヒト抗体を取得してもよい （国際特許出願公開番号 W092- 03918、 W093-2 227、 W094-02602, W094- 25585、 W096- 33735および W096- 34096参照) 。  Furthermore, a transgenic animal having a human antibody gene repertoire is immunized with a protein serving as an antigen, a protein-expressing cell or a lysate thereof to obtain antibody-producing cells, and a hybridoma obtained by fusing the antibody-producing cells with myeloma cells is obtained. May be used to obtain a human antibody against the protein (see International Patent Application Publication Nos. W092-03918, W093-2227, W094-02602, W094-25585, W096-33735, and W096-34096).
ハイプリ ド一マを用いて抗体を産生する以外に、 抗体を産生する感作リンパ 球等の免疫細胞を癌遺伝子 （oncogene )により不死化させた細胞を用いてもよ い o  In addition to producing antibodies using hybridomas, cells in which immune cells such as sensitized lymphocytes that produce antibodies are immortalized with oncogenes may be used.o
このように得られたモノクローナル抗体はまた、 遺伝子組換え技術を用いて 産生させた組換え型抗体として得ることができる （例えば、 Borrebaeck, C. A. K. and Larrick, J. W. , THERAPEUTIC MONOCLONAL ANTIBOD IES, Publ ished i n the United Kingdom by MACMILLAN PUBL ISHERS LTD, 1990 参照）。 組換え型 抗体は、 それをコードする D N Aをハイプリ ドーマ又は抗体を産生する感作リ ンパ球等の免疫細胞からクローニングし、 適当なベクターに組み込んで、 これ を宿主に導入し産生させる。 本発明は、 この組換え型抗体を包含する。  The monoclonal antibody thus obtained can also be obtained as a recombinant antibody produced using a gene recombination technique (for example, Borrebaeck, CAK and Larrick, JW, THERAPEUTIC MONOCLONAL ANTIBOD IES, Published in the United Kingdom by MACMILLAN PUBL ISHERS LTD, 1990). The recombinant antibody is produced by cloning DNA encoding the antibody from an immune cell such as a hybridoma or a sensitized lymphocyte producing the antibody, incorporating the DNA into an appropriate vector, and introducing the vector into a host. The present invention includes this recombinant antibody.
本発明の抗体は、 本発明の蛋白質に結合するかぎり、 その抗体断片や抗体修 飾物であってよい。 例えば、 抗体断片としては、 Fab、 F ( ab' )2、 Fv又は H鎖と L 鎖の Fvを適当なリンカーで連結させたシングルチエイン Fv( scFv ) (Huston, J . S. et al .， Proc . Natl . Acad. Sci . U. S.A. ( 1988 ) 85 , 5879- 5883 )が挙げら れる。 具体的には、 抗体を酵素、 例えば、 パパイン、 ペプシンで処理し抗体断 片を生成させるか、 又は、 これら抗体断片をコードする遺伝子を構築し、 これ を発現ベクターに導入した後、 適当な宿主細胞で発現させる （例えば、 Co， M. S. et al . ， J. Immunol . ( 1994) 152, 2968-2976 ； Better, M. and Horwitz, A. H. ， Methods Enzymol . ( 1989 ) 178, 476-496 ； Pluckthun, A. and Skerr a, Aリ Methods Enzymol . ( 1989 ) 178, 497-515 ； Lamoyi , E. ， Methods Enzy mol . ( 1986 ) 121, 652-663 ； Rousseaux, J. et al . ， Methods Enzymol . ( 198 6 ) 121 , 663-669 ； Bird, R. E. and Walker, B. W. , Trends Biotechnol . ( 1 991 ) 9， 132-137参照）。 The antibody of the present invention may be an antibody fragment or an antibody modification thereof as long as it binds to the protein of the present invention. For example, as an antibody fragment, Fab, F (ab ') 2, Fv, or a single chain Fv (scFv) in which an Fv of an H chain and an L chain are linked by an appropriate linker (Huston, J. S. et al., Natl. Acad. Sci. USA (1988) 85, 5879-5883). Specifically, the antibody is treated with an enzyme, for example, papain or pepsin, to generate an antibody fragment, or a gene encoding these antibody fragments is constructed and, after introducing this into an expression vector, an appropriate host Expressed in cells (eg, Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669. Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137).
抗体修飾物として、 ポリエチレングリコール（PEG)等の各種分子と結合した 抗体を使用することもできる。 本発明の 「抗体」 にはこれらの抗体修飾物も包 含される。 このような抗体修飾物を得るには、 得られた抗体に化学的な修飾を 施すことによって得ることができる。 これらの方法はこの分野において既に確 立されている。  As the modified antibody, an antibody bound to various molecules such as polyethylene glycol (PEG) can also be used. The “antibody” of the present invention also includes these modified antibodies. Such a modified antibody can be obtained by subjecting the obtained antibody to chemical modification. These methods have already been established in this field.
また、 本発明の抗体は、 公知の技術を使用して非ヒト抗体由来の可変領域と ヒト抗体由来の定常領域からなるキメラ抗体又は非ヒト抗体由来の CDR (相補性 決定領域） とヒト抗体由来の FR (フレームワーク領域） 及び定常領域からなる ヒト型化抗体として得ることができる。  In addition, the antibody of the present invention can be prepared by using a chimeric antibody comprising a non-human antibody-derived variable region and a human antibody-derived constant region or a non-human antibody-derived CDR (complementarity determining region) and a human antibody-derived antibody using known techniques. Can be obtained as a humanized antibody consisting of the FR (framework region) and constant region.
前記のように得られた抗体は、 均一にまで精製することができる。 本発明で 使用される抗体の分離、 精製は通常の蛋白質で使用されている分離、 精製方法 を使用すればよく、 何ら限定されるものではない。 上記で得られた抗体の濃度 測定は吸光度の測定又は酵素結合免疫吸着検定法（Enzyme- l inked immunosorbe nt assay； ELISA) 等により行うことができる。  The antibody obtained as described above can be purified to homogeneity. The separation and purification of the antibody used in the present invention may be performed by the separation and purification methods used for ordinary proteins, and is not limited at all. The concentration of the antibody obtained as described above can be measured by measuring absorbance, enzyme-linked immunosorbent assay (ELISA), or the like.
また、 本発明の抗体の抗原結合活性を測定する方法として、 ELISA、 EIA (酵 素免疫測定法） 、 RIA (放射免疫測定法） あるいは蛍光抗体法を用いることがで きる。例えば、 ELISAを用いる場合、 本発明の抗体を固相化したプレートに本発 明の蛋白質を添加し、 次いで目的の抗体を含む試料、 例えば、 抗体産生細胞の 培養上清や精製抗体を加える。 酵素、 例えばアルカリフォスファタ一ゼ等で標 識した抗体を認識する二次抗体を添加し、 プレートをインキュベーション、 洗 浄した後、 P-二トロフエニル燐酸などの酵素基質を加えて吸光度を測定するこ とで抗原結合活性を評価することができる。 蛋白質として蛋白質の断片、 例え ばその C末端からなる断片あるいは N末端からなる断片を使用してもよい。 本発 明の抗体の活性評価には、 BIAcore(Pharmacia製）を使用することができる。 これらの手法を用いることにより、 本発明の抗体と試料中に含まれる本発明 の蛋白質が含まれると予想される試料とを接触せしめ、 前記抗体と蛋白質との 免疫複合体を検出又は測定することから成る本発明の蛋白質の検出又は測定方 法を実施することができる。 As a method for measuring the antigen-binding activity of the antibody of the present invention, ELISA, EIA (enzyme immunoassay), RIA (radioimmunoassay) or a fluorescent antibody method can be used. For example, when ELISA is used, the protein of the present invention is added to a plate on which the antibody of the present invention is immobilized, and then a sample containing the target antibody, for example, a culture supernatant of antibody-producing cells or a purified antibody is added. Add a secondary antibody that recognizes the antibody labeled with an enzyme, for example, alkaline phosphatase, and incubate and wash the plate. After purification, antigen binding activity can be evaluated by adding an enzyme substrate such as P-ditrophenyl phosphate and measuring the absorbance. As the protein, a protein fragment, for example, a C-terminal fragment or an N-terminal fragment thereof may be used. BIAcore (Pharmacia) can be used to evaluate the activity of the antibody of the present invention. By using these techniques, the antibody of the present invention is brought into contact with a sample expected to contain the protein of the present invention contained in the sample, and an immune complex of the antibody and the protein is detected or measured. Or the method of detecting or measuring the protein of the present invention comprising:
本発明の蛋白質の検出又は測定方法は、 蛋白質を特異的に検出又は測定する ことができるため、 蛋白質を用いた種々の実験等に有用である。  Since the protein detection or measurement method of the present invention can specifically detect or measure a protein, it is useful for various experiments and the like using proteins.
本発明は、 配列番号： 2、 配列番号： 4、 配列番号： 6、 配列番号： 8、 配 列番号： 2 0及び配列番号： 2 2 ら 2 7のいずれか一つに示される塩基配列 からなる D N Aまたは該 D N Aと相補的な D N Aと特異的にハイブリダィズし、 少なくとも 1 5塩基の鎖長を有する D N Aを包含する。 すなわち、 本発明の蛋 白質をコ一ドする D N A又は該 D N Aと相補的な D N Aと選択的にハイプリダ ィズし得るプローブ、 ヌクレオチド又はヌクレオチド誘導体、 例えばアンチセ ンスオリゴヌクレオチド、 リボザィム等が含まれる。  The present invention relates to a nucleotide sequence represented by any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20 and SEQ ID NO: 22 to 27 DNA that specifically hybridizes with DNA or DNA complementary to the DNA and has a chain length of at least 15 bases. That is, probes, nucleotides or nucleotide derivatives capable of selectively hybridizing DNA encoding the protein of the present invention or DNA complementary to the DNA, such as antisense oligonucleotides and ribozymes, are included.
本発明は、 例えば、 配列番号： 2、 配列番号： 4、 配列番号： 6、 配列番号： 8、 配列番号： 2 0及び配列番号 ： 2 2から 2 7のいずれか一つに示される塩 基配列中のいずれかの箇所にハイプリダイズするアンチセンスオリゴヌクレオ チドを含む。 このアンチセンスオリゴヌクレオチドは、 好ましくは配列番号： 2、 配列番号： 4、 配列番号： 6、 配列番号： 8、 配列番号： 2 0及び配列番 号： 2 2から 2 7のいずれか一つに示される塩基配列中の連続する少なくとも 1 5個以上のヌクレオチドに対するアンチセンスオリゴヌクレオチドである。 さらに好ましくは、 前記連続する少なくとも 1 5個以上のヌクレオチドが翻訳 開始コ ドンを含む、 前記のアンチセンスオリゴヌクレオチドである。 ό The present invention relates to, for example, a base represented by any one of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20 and SEQ ID NO: 22 to 27 Includes antisense oligonucleotides that hybridize anywhere in the sequence. This antisense oligonucleotide preferably has SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 20 and any one of SEQ ID NOs: 22 to 27. It is an antisense oligonucleotide to at least 15 or more consecutive nucleotides in the nucleotide sequence shown. More preferably, the antisense oligonucleotide described above, wherein the continuous at least 15 or more nucleotides include a translation initiation codon. ό
アンチセンスオリゴヌクレオチドとしては、 それらの誘導体や修飾体を使用 することができる。 このような修飾体として、 例えばメチルホスホネート型又 はェチルホスホネート型のような低級アルキルホスホネート修飾体、 ホスホロ チォエート修飾体又はホスホロアミデ一ト修飾体等が挙げられる。 As the antisense oligonucleotide, derivatives and modifications thereof can be used. Examples of such modifications include lower alkylphosphonate modifications such as methylphosphonate type or ethylphosphonate type, phosphorothioate modifications and phosphoramidite modifications.
ここでいう 「アンチセンスオリゴヌクレオチド」 とは、 DNA又は mRNA の所定の領域を構成するヌクレオチドに対応するヌクレオチドが全て相補的で あるもののみならず、 DNA又は mRNAとオリゴヌクレオチドとが配列番 号： 1に示される塩基配列に選択的に安定にハイブリダィズできる限り、 1又は 複数個のヌクレオチドのミスマッチが存在していてもよい。  As used herein, the term “antisense oligonucleotide” includes not only those in which all the nucleotides corresponding to the nucleotides constituting the predetermined region of DNA or mRNA are complementary, but also those in which DNA or mRNA and the oligonucleotide are sequence numbers: A mismatch of one or more nucleotides may exist as long as it can selectively and stably hybridize to the base sequence shown in 1.
選択的に安定にハイプリダイズするとは、 通常のハイブリダィゼーシヨン条 件下、 好ましくはストリンジェントなハイブリダイゼ一ション条件下で、 他の 蛋白質をコ一ドする DN Aとのクロスハイプリダイゼーシヨンが有意に生じな いことを意味する。 このような DNAとしては、 少なくとも 15個の連続したヌ クレオチド配列領域で、 少なくとも 70%、 好ましくは少なくとも 80%、 より好ま しくは 90%、 さらに好ましくは 95%以上の塩基配列上の相同性を有するものを示 す。 なお、 相同性を決定するためのアルゴリズムは本明細書に記載したものを 使用すればよい。 このような DN Aは、 後述の実施例に記載するように本発明 の蛋白質をコ一ドする DN Aを検出若しくは単離するためのプローブとして、 又は増幅するためのプライマーとして有用である。  Selectively and stably hybridizing is defined as cross-hybridization with DNA encoding other proteins under normal hybridization conditions, preferably under stringent hybridization conditions. Does not occur significantly. Such DNAs should have at least 15 contiguous nucleotide sequence regions with at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95% or more nucleotide sequence homology. Show what you have. The algorithm for determining homology may use the algorithm described in this specification. Such a DNA is useful as a probe for detecting or isolating a DNA encoding the protein of the present invention, or as a primer for amplifying, as described in Examples below.
本発明のアンチセンスオリゴヌクレオチド誘導体は、 本発明の蛋白質の産生 細胞に作用して、 該蛋白質をコ一ドする DN A又は mRNAに結合することに より、 その転写又は翻訳を阻害したり、 mRNAの分解を促進したりして、 本 発明の蛋白質の発現を抑制することにより、 結果的に本発明の蛋白質の作用を 抑制する効果を有する。  The antisense oligonucleotide derivative of the present invention acts on cells producing the protein of the present invention to inhibit transcription or translation of the protein by binding to DNA or mRNA encoding the protein, By suppressing the expression of the protein of the present invention, for example, by promoting the degradation of the protein, the effect of suppressing the action of the protein of the present invention is obtained.
本発明のアンチセンスオリゴヌクレオチド誘導体は、 それらに対して不活性 な適当な基剤と混和して塗布剤、 パップ剤等の外用剤とすることができる。 また、 必要に応じて、 賦形剤、 等張化剤、 溶解補助剤、 安定化剤、 防腐剤、 無痛化剤等を加えて錠剤、 散財、 顆粒剤、 カプセル剤、 リポソ一ムカプセル剤、 注射剤、 液剤、 点鼻剤など、 さらに凍結乾燥剤とすることができる。 これらは 常法にしたがって調製することができる。 The antisense oligonucleotide derivative of the present invention can be used as an external preparation such as a liniment or a poultice by mixing with a suitable base material which is inactive against the derivative. If necessary, excipients, isotonic agents, solubilizing agents, stabilizers, preservatives, soothing agents, etc. are added to tablets, splinters, granules, capsules, liposomal capsules, and injections. Agents, solutions, nasal drops, etc., and further can be lyophilized. These can be prepared according to a conventional method.
本発明のアンチセンスオリゴヌクレオチド誘導体は患者の患部に直接適用す るか、 又は血管内に投与するなどして結果的に患部に到達し得るように患者に 適用する。 さらには、 持続性、 膜透過性を高めるアンチセンス封入素材を用い ることもできる。 例えば、 リボソーム、 ポリ- L-リジン、 リビッ ド、 コレステロ —ル、 リポフヱクチン又はこれらの誘導体が挙げられる。  The antisense oligonucleotide derivative of the present invention is applied directly to the affected area of the patient, or is applied to the patient so that it can reach the affected area as a result of intravenous administration or the like. Furthermore, an antisense-encapsulated material that enhances durability and membrane permeability can be used. For example, ribosome, poly-L-lysine, ribid, cholesterol, lipofectin or derivatives thereof can be mentioned.
本発明のアンチセンスオリゴヌクレオチド誘導体の投与量は、 患者の状態に 応じて適宜調整し、 好ましい量を用いることができる。 例えば、 0. 1〜100mg/k g好ましくは 0. 1〜50mg/kgの範囲で投与することができる。  The dosage of the antisense oligonucleotide derivative of the present invention can be appropriately adjusted according to the condition of the patient, and a preferred amount can be used. For example, it can be administered in the range of 0.1 to 100 mg / kg, preferably 0.1 to 50 mg / kg.
本発明のアンチセンスオリゴヌクレオチドは本発明の蛋白質の発現を阻害し、 したがって本発明の蛋白質の生物学的活性を抑制することにおいて有用である また、 本発明のアンチセンスオリゴヌクレオチドを含有する発現阻害剤は、 本 発明の蛋白質の生物学的活性を抑制することが可能である点で有用である。 図面の簡単な説明  The antisense oligonucleotide of the present invention inhibits the expression of the protein of the present invention, and is therefore useful in suppressing the biological activity of the protein of the present invention. Expression inhibition containing the antisense oligonucleotide of the present invention The agent is useful in that it can suppress the biological activity of the protein of the present invention. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 AC002303配列中に唯一存在するプローブ配列 40952- 40966を含む 409 52- 40966の 180塩基を質問式 （query) とした BlastX検索による結果を示す模式 図である。 「#」 ： NR8についてのみ番号はヌクレオチド数にて表示した。 NR8 配列に付したアンダーラインはェクソンに相当する部分を示す。 他のアンダー ラインで示した配列は、 同一アミノ酸を示す。  FIG. 1 is a schematic diagram showing the result of a BlastX search using 180 bases of 40952-40966 including the only probe sequence 40952-40966 in the AC002303 sequence as a query. “#”: The number is indicated by nucleotide number only for NR8. An underline attached to the NR8 sequence indicates a portion corresponding to exon. Other underlined sequences indicate identical amino acids.
図 2は、 AC002303配列中に唯一存在するプローブ配列 40952- 40966を含む 409 52-40966の 180塩基を中心に 5'、 3'両方向に 180塩基ずつ B lastXスキャンした結 果を示す模式図である。 図 3は、 ヒト胎児肝、 骨格筋 c DNAを錶型として SN1/AS1、 SN1/AS2、 SN2/ ASK SN2/AS2のプライマ一の組み合わせで RT-PCT法にて増幅した結果を電気泳 動した時の図を示す。 Figure 2 is a schematic diagram showing the result of B lastX scan of 180 bases in both 5 'and 3' directions, centering on 180 bases of 409 52-40966 including the only probe sequence 40952-40966 in the AC002303 sequence. . Figure 3 shows the results of RT-PCT amplification using human fetal liver and skeletal muscle cDNA as type III, amplified by a combination of SN1 / AS1, SN1 / AS2, and SN2 / ASK SN2 / AS2 primers. Figure shows the time.
図 4は、 ヒト胎児肝 c DN Aを錄型として 5'- RACE法、 3' -RACE法を実施した 結果を電気泳動した時の図を示す。  FIG. 4 shows the results of electrophoresis of the results of performing the 5′-RACE method and the 3′-RACE method using human fetal liver cDNA as type II.
図 5は、 NR8ひの cDNAの塩基配列及びアミノ酸配列を示す図である。 RT- PCRに用いたプライマーの位置を矢印で示した。 5'側から順に、 SN 1 ( 798-827 )、 SN2(894-923), AS2(1055-1026)、 AS1(1127-1098)である。但し AS1については 5 '端の 2塩基は CTではなく、 ゲノム由来の ACを用いた。  FIG. 5 shows the nucleotide sequence and amino acid sequence of the cDNA of NR8. The positions of the primers used for RT-PCR are indicated by arrows. In order from the 5 'side, they are SN1 (798-827), SN2 (894-923), AS2 (1055-1026), and AS1 (1127-1098). However, for AS1, the genome-derived AC was used instead of CT for the two bases at the 5 'end.
図 6は、 NR8ひの c D N Aの塩基配列及びァミノ酸配列の図 5の続きを示す図 である。  FIG. 6 is a continuation of FIG. 5 showing the nucleotide sequence and amino acid sequence of cDNA of NR8.
図 7は、 NR8 ?の cDN Aの塩基配列及びアミノ酸配列を示す図である。二つ の可能なオープンリーディングフレーム （0RF) を示した。  FIG. 7 shows the nucleotide sequence and amino acid sequence of NR8? CDNA. Two possible open reading frames (0RF) are shown.
図 8は、 の cDN Aの塩基配列及びアミノ酸配列を示す図 7の続きの図 である。  FIG. 8 is a continuation of FIG. 7 showing the nucleotide sequence and amino acid sequence of the cDNA of FIG.
図 9は、 NR8ァの c D N Aの塩基配列及びアミノ酸配列を示す図である。選択 的スプライシングにより挿入される 177アミノ酸を下線で示した。  FIG. 9 is a diagram showing the nucleotide sequence and amino acid sequence of cDNA of NR8. The 177 amino acids inserted by alternative splicing are underlined.
図 1 0は、 NR8ァの c D N Aの塩基配列及びァミノ酸配列を示す図 9の続きの 図である。選択的スプライシングにより挿入される 177アミノ酸に下線を施した c 図 1 1は、 NR87の cDNAの塩基配列及びアミノ酸配列を示す図 1 0の続き の図である。 FIG. 10 is a continuation of FIG. 9 showing the nucleotide sequence and the amino acid sequence of the NR8 cDNA. C Figure 1 1 which underlined 177 amino acids inserted by alternative splicing are continuation of FIG. 1 0 shows the nucleotide sequence and amino acid sequence of the cDNA of NR87.
図 1 2は、 NR8の各臓器における発現をノーザンプロッティングにより解析し た結果を示す図である。  FIG. 12 is a diagram showing the results of analyzing the expression of NR8 in each organ by Northern plotting.
図 1 3は、 NR8遺伝子の構造を模式的に示す図である。他の繰り返しには、 （C A)n， (CAGA)n, (TGGA)n, (CATA)n, (TA)n, (GA)n, (GGAA)n, (CATG)n, (GAM) n, MSTA, AT-rich, LT1A1, LINE2, FLAM— C, MER63A, MSTBを含む。 図 1 4は、 発現ベクターにおいて構築した発現可能な蛋白質の構造模式図を 示す。 FIG. 13 is a diagram schematically showing the structure of the NR8 gene. Other iterations include (CA) n, (CAGA) n, (TGGA) n, (CATA) n, (TA) n, (GA) n, (GGAA) n, (CATG) n, (GAM) n , MSTA, AT-rich, LT1A1, LINE2, FLAM-C, MER63A, MSTB. FIG. 14 shows a schematic structural diagram of an expressible protein constructed in an expression vector.
図 1 5は、 ヒ ト NR8 プライマ一セッ トをマウス cDNAライブラリ一に対して用 いた、 交叉 PCR の結果を示す。 サイズマーカーとして 100 bp DNA Ladder (NEB #323- 1L )を用いた。  FIG. 15 shows the results of cross-PCR using a human NR8 primer set against a mouse cDNA library. A 100 bp DNA Ladder (NEB # 323-1L) was used as a size marker.
図 1 6は、 ヒ ト及びマウス NR8 3のアミノ酸配列を比較した結果を示す。両者 がー致するアミノ酸配列に色塗りを施した。 また配列中、 他のへモポェチン受 容体に保存されたシスティン残基を太字で記した。  FIG. 16 shows the results of comparing the amino acid sequences of human and mouse NR83. The amino acid sequence where the two match was colored. In the sequence, cysteine residues conserved in other hemopoietin receptors are shown in bold.
図 1 7は、 ヒ 卜及びマウス NR8ァのアミノ酸配列を比較した結果を示す。両者 がー致するアミノ酸配列に色塗りを施した。 また配列中、 他のへモポェチン受 容体に保存されたシスティン残基、 及び WSXWS-Boxを太字で記した。  FIG. 17 shows the results of comparing the amino acid sequences of human and mouse NR8a. The amino acid sequence where the two match was colored. In the sequence, cysteine residues conserved in other hemopoietin receptors and WSXWS-Box are shown in bold.
図 1 8は、 RT-PCR法によって各マウス臓器における NR8遺伝子の発現様態を解 析した結果を示す。 サイズマ一カーとして 100 bp DNA Ladder (NEB#323- 1L )を 用い、 レーンの両端に示した。何れの臓器においても 320 bp の標的遺伝子が検 出されている。  FIG. 18 shows the results of analyzing the expression of the NR8 gene in each mouse organ by RT-PCR. 100 bp DNA Ladder (NEB # 323-1L) was used as a size marker, and indicated at both ends of the lane. A 320 bp target gene was detected in all organs.
図 1 9は、 ノーザンブロッテイング法によって、 各マウス臓器における NR8 遺伝子の発現様態を解析した結果を示す （左） 。 精巣においてのみ、 約 4.2 kb の転写産物が強く検出された。陽性対照として同一プロッ トのマウスべ一夕ァ クチンの検出も実施した （右） 。 発明を実施するための最良の形態  Figure 19 shows the results of analyzing the expression of the NR8 gene in each mouse organ by Northern blotting (left). Only in the testis, a transcript of about 4.2 kb was strongly detected. As a positive control, we also detected mouse whole actin of the same plot (right). BEST MODE FOR CARRYING OUT THE INVENTION
次に、 本発明を実施例によりさらに具体的に説明するが、 本発明は下記実施 例に限定されるものではない。  Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples.
[実施例 1 ] 2段階 Blast検索  [Example 1] Two-stage Blast search
Trp-Ser-Xaa-Trp-Serモチーフをコードするオリゴヌクレオチドとして tggag ( t/c )nnntggag(t/c ) (nは任意の塩基） からなる 256通りのプローブ配列をデザ O 99/67290 ^ PC冒 9画 51 As an oligonucleotide encoding the Trp-Ser-Xaa-Trp-Ser motif, 256 probe sequences consisting of tggag (t / c) nnntggag (t / c) (n is an arbitrary base) are designed. O 99/67290 ^ PC first 9 strokes 51
インした。 この配列により EPO受容体、 TP0受容体及びマウス IL6受容体を除く殆 ど全ての既知へモポェチン受容体が検出可能である。 個々の配列を質問式 （qu ery)として GenBankの nrデ一夕べ一スを BlastN(Advanced BlastN 2.0.4)プログ ラムを用いて検索した。 検索のパラメ一夕は期待値を 100とした以外は def auU 値 (Descriptions=100、 Alignments=100) を用いた。 In. This sequence allows the detection of almost all known hemopoietin receptors except the EPO receptor, TP0 receptor and mouse IL6 receptor. Each sequence was used as a query and the nr database of GenBank was searched using the BlastN (Advanced BlastN 2.0.4) program. For the search parameters, def auU values (Descriptions = 100, Alignments = 100) were used except that the expected value was set to 100.
この一次検索の結果、約 500個のプロ -ブ配列と完全にマッチするクローンが 得られたので、 このうちヒトゲノム由来クローン （コスミ ド、 BAC及び PAC) に ついて、 プローブ配列をほぼ中央に含む 180塩基の配列を切り出した。次にこの 180塩基からなる配列を質問式 (query) として BlastX (Advanced BlastX 2.0. 4) プログラムを用いて再度 nrデ一夕べ一スを検索することにより、プロ一ブ配 列周辺のアミノ酸配列の既知へモポェチン受容体との相同性を検索した。  As a result of this primary search, clones completely matching approximately 500 probe sequences were obtained. Of these, clones derived from the human genome (cosmid, BAC, and PAC) containing the probe sequence at almost the center 180 The base sequence was cut out. Next, the sequence consisting of 180 bases was used as a query and the nr database was searched again using the BlastX (Advanced BlastX 2.0.4) program to find the amino acid sequence around the probe sequence. Homology with known hemopoietin receptors was searched.
検索のパラメ一夕は、通常、期待値を 100とした以外は default値を用いたが、 しばしば極めて多数のヒット （高度反復配列である Aluサブフアミリーに起因） が得られた場合に既知へモポェチン受容体へのヒッ トを観測することが困難で あったことから、 この様な場合は感度を最大限とするため、 「Expect=1000、 D escriptions=500、 Align腿 ts=500」 の値を用いた。  Normally, default values were used for the search parameters except that the expected value was set to 100. However, often a very large number of hits (attributable to the highly repetitive sequence Alu subfamily) were obtained. Since it was difficult to observe the hits to the receptor, in such cases, the values of “Expect = 1000, Descriptions = 500, Align thighs = 500” were used to maximize the sensitivity. Using.
BlastXによる 2次検索の結果、 28のクローンが 1個以上の既知へモポェチン 受容体をヒッ 卜した （表 1から表 8 ) 。 As a result of a secondary search using BlastX, 28 clones hit one or more known hemopoietin receptors (Tables 1 to 8).
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Figure imgf000040_0001
Figure imgf000040_0001
プローブ Xaa ァクセシヨン番号 ヒットした位 S 座位 blastx (expect=100) Probe Xaa accession number Hit position S Locus blastx (expect = 100)
TTGGAGTTTCTGGAGC Phe AC002112 68699 tggagtttctggagc 68685 6 IgHv, MYD116  TTGGAGTTTCTGGAGC Phe AC002112 68699 tggagtttctggagc 68685 6 IgHv, MYD116
TGGAGCGGCTGGAGC Gly U89336 35829 tggagcggctggagc 35815 6p21 myosin HC, cep250,  TGGAGCGGCTGGAGC Gly U89336 35829 tggagcggctggagc 35815 6p21 myosin HC, cep250,
TQGAGCGTCTGGAGC Val U53588 3558 tggagcgtctggagc 3572 6p21.3 ring finger, BRCAl  TQGAGCGTCTGGAGC Val U53588 3558 tggagcgtctggagc 3572 6p21.3 ring finger, BRCAl
TGGAGTGCATGGAGT Ala Z98744 38358 tgga tgcatg^agt 38344 6p21.3-22.3 Alu, AD7c-NTP  TGGAGTGCATGGAGT Ala Z98744 38358 tgga tgcatg ^ agt 38344 6p21.3-22.3 Alu, AD7c-NTP
TGGAGTTGCTGGAGT Cys AL009031 104325 tggagttgctgsagt 104311 6p22.3-24.1 ACC synthase  TGGAGTTGCTGGAGT Cys AL009031 104325 tggagttgctgsagt 104311 6p22.3-24.1 ACC synthase
TGGAGTGTCTGGAGT Val AL008729 21325 tggagtgtctggagt 21339 6p24 E1A, DUB-2  TGGAGTGTCTGGAGT Val AL008729 21325 tggagtgtctggagt 21339 6p24 E1A, DUB-2
TGGAGTTGTTGGAGT Cys Z98755 69825 tggagttgttggagt 69811 6ql6.1-21 dynein  TGGAGTTGTTGGAGT Cys Z98755 69825 tggagttgttggagt 69811 6ql6.1-21 dynein
TGGAGCTTCTGGAGC Phe Z98172 36554 tggagcttctggagc 35540 6q21 HGXPRT  TGGAGCTTCTGGAGC Phe Z98172 36554 tggagcttctggagc 35540 6q21 HGXPRT
TGGAGCAGGTGGAGC Arg Z97989 79116 tggagcaggtggagc 79102 6q21-22 syn fyn, slk, yes, arc  TGGAGCAGGTGGAGC Arg Z97989 79116 tggagcaggtggagc 79102 6q21-22 syn fyn, slk, yes, arc
TGGAGCTAATGGAGT *** Z95326 16562 tggagctaatggagt 16576 6q22.1-6q22.33 tyrosinase  TGGAGCTAATGGAGT *** Z95326 16562 tggagctaatggagt 16576 6q22.1-6q22.33 tyrosinase
1 D 1D
TGGAGCTCTTGGAQC Ser Z98049 25800 tggagctcttggagc 25786 6q26-q27 collagen, AT3, ClQb TGGAGCTCTTGGAQC Ser Z98049 25800 tggagctcttggagc 25786 6q26-q27 collagen, AT3, ClQb
TGGAGCTCCTGGAGT Ser AC003090 22068 tggagctectggagt 22082 7p l5 ICE  TGGAGCTCCTGGAGT Ser AC003090 22068 tggagctectggagt 22082 7p l5 ICE
TGGAGTATATGGAGC Be AC004744 22740 tggagtatatggagc 22754 7pl5-p21 TSH-R, RNABP  TGGAGTATATGGAGC Be AC004744 22740 tggagtatatggagc 22754 7pl5-p21 TSH-R, RNABP
TGGAGTAGCTGGAGC Ser AC004485 86356 tggag agctggagc 86370 7pl5-p2l Hnv 2 4 mTT I ITW *、  TGGAGTAGCTGGAGC Ser AC004485 86356 tggag agctggagc 86370 7pl5-p2l Hnv 24 mTT I ITW *,
TGGAGTCTTTGGAGT Leu AC004141 3130 tggagtctttggagt 3144 7p21-p22 polyprotein  TGGAGTCTTTGGAGT Leu AC004141 3130 tggagtctttggagt 3144 7p21-p22 polyprotein
TGGAGCAGATGGAGC Arg AC004548 62876 tggagcagatggagc 62862 7qll.23-q21.1 NCAM  TGGAGCAGATGGAGC Arg AC004548 62876 tggagcagatggagc 62862 7qll.23-q21.1 NCAM
TGGAGCAACTGGAGT Asn AC002456 69500 tggagcaactggagt 69514 7q21 glycoprotein A  TGGAGCAACTGGAGT Asn AC002456 69500 tggagcaactggagt 69514 7q21 glycoprotein A
TGGAGTAACTGGAGT Asn AC000064 9170 tggagtaactggagt 9184 7q21-22 GA3PD  TGGAGTAACTGGAGT Asn AC000064 9170 tggagtaactggagt 9184 7q21-22 GA3PD
TGGAGTTATTGGAGT Tyx AC003085 87341 tggag tattggag^ 87355 7q21-22 Nmyc, FGFR  TGGAGTTATTGGAGT Tyx AC003085 87341 tggag tattggag ^ 87355 7q21-22 Nmyc, FGFR
TGGAGTTGTTGGAGT Cys AC000119 65235 tggagttgttggagt 65221 7q21-7q22 FVIII, T poIII  TGGAGTTGTTGGAGT Cys AC000119 65235 tggagttgttggagt 65221 7q21-7q22 FVIII, T poIII
TGGAGTTGTTGGAGT Cys AC002458 44435 tggagttgttggaKt 44421 7q21-q22 telomerase, NFAT  TGGAGTTGTTGGAGT Cys AC002458 44435 tggagttgttggaKt 44421 7q21-q22 telomerase, NFAT
TGGAGTACATGGAGC Thr AC000059 9977 tgga acatggagc 9963 7q21-7q22 Alu, Notch4 TGGAGTACATGGAGC Thr AC000059 9977 tgga acatggagc 9963 7q21-7q22 Alu, Notch4
ァク ンヨン番号 ヒットした位 B 座位 blastx (expect=100) Function number Hit position B Sitting position blastx (expect = 100)
TTGGAGTATTTGGAGT He AC002384 52216 tggagtatttggagt 52202 7q22 pol, RHR(anot pr framet TTGGAGTATTTGGAGT He AC002384 52216 tggagtatttggagt 52202 7q22 pol, RHR (anot pr framet
TGGAGCAGCTGGAGT Ser AC004522 55291 tggagc&gctggagt 55277 1 hemoglobin beta  TGGAGCAGCTGGAGT Ser AC004522 55291 tggagc & gctggagt 55277 1 hemoglobin beta
TGGAGTGTTTGGAGT Val AC002466 43273 tggagtgtttggagt 43287 7q31 ryanodine receptor, mTPO TGGAGTGTTTGGAGT Val AC002466 43273 tggagtgtttggagt 43287 7q31 ryanodine receptor, mTPO
TGGAGTGGCTGGAGC Gly AC002543 112948 tg aetggctggagc 112962 7q31.2 EOF, P-selectin TGGAGTGGCTGGAGC Gly AC002543 112948 tg aetggctggagc 112962 7q31.2 EOF, P-selectin
TGGAGCTGATGGAGC * G G** AC000061 79564 tggagctgatggagc 79550 7q31.2 laminin Bl, tubulin  TGGAGCTGATGGAGC * GG ** AC000061 79564 tggagctgatggagc 79550 7q31.2 laminin Bl, tubulin
TGGAGTTTTTGGAGT Phe AC000125 13750 tggagtttttggaft 13736 7q31.3 pl50  TGGAGTTTTTGGAGT Phe AC000125 13750 tggagtttttggaft 13736 7q31.3 pl50
TGGAGTTGTTGGAGT Cys AC002498 20166 tggagttg^tggagt 20162 7q31.3 IL3Rhrnpposit.Rl  TGGAGTTGTTGGAGT Cys AC002498 20166 tggagttg ^ tggagt 20162 7q31.3 IL3Rhrnpposit.Rl
TGGAGCGGGTGGAGC Gly U66059 158491 tggagcgggtggagc 158477 7q35(TcRb) properdin  TGGAGCGGGTGGAGC Gly U66059 158491 tggagcgggtggagc 1558477 7q35 (TcRb) properdin
TGGAGCATTTGGAGC He AC003109 4761 tggagcatttggagc 4775 7q36 CD2, HOX-2.6  TGGAGCATTTGGAGC He AC003109 4761 tggagcatttggagc 4775 7q36 CD2, HOX-2.6
TGGAGTTATTGGAGT Tyr AF027390 174448 tggag^tattggagt 174434 7q tel IkB, V2R CO TGGAGTTATTGGAGT Tyr AF027390 174448 tggag ^ tattggagt 174434 7q tel IkB, V2R CO
TGGAGCATATGGAGT lie (:0020.r)2 28882 tggagcatatggagt 28896 9p22 myosin VIIA, | OSMIil) TGGAGCATATGGAGT lie (:. 0020 r) 2 28882 tggagcatatggagt 28896 9p22 myosin VIIA, | OSMIil)
TGGAGCAACTGGAGT Asn AC001643 27345 tggagcaactggagt 27331 9q34 hoxl.4, gastrinR TGGAGCAACTGGAGT Asn AC001643 27345 tggagcaactggagt 27331 9q34 hoxl.4, gastrinR
;綱 疆，躍  ; Rinjiang, Leap
TGGAGCGGATGGAGC AC000396 16394 tggagcggatggagc 16380 9q34 vWf, laminin a3  TGGAGCGGATGGAGC AC000396 16394 tggagcggatggagc 16380 9q34 vWf, laminin a3
TGGAGTGAGTGGAGT U73649 168501 a^tgaKtgtraKt 16836 11 zinc finger TGGAGTGAGTGGAGT U73649 168501 a ^ tgaKtgtraKt 16836 11 zinc finger
TGGAGTGCCTGGAGT Ala U73629 31027 tggag^gcctggag^ 31041 11 Alu, gp2b, BCGF-12 TGGAGTGCCTGGAGT Ala U73629 31027 tggag ^ gcctggag ^ 31041 11 Alu, gp2b, BCGF-12
TGGAGTCCCTGGAGC Pro U73643 14550 tgga tccctggagc 14564 11 reverse transcriptase TGGAGTCCCTGGAGC Pro U73643 14550 tgga tccctggagc 14564 11 reverse transcriptase
TGGAGCAACTGGAGC Asn FJtlHHIIti 65621 tggagcaactggagc 65635 lip 15.5 Nasopressin. R,| OSMK TGGAGCAACTGGAGC Asn FJtlHHIIti 65621 tggagcaactggagc 65635 lip 15.5 Nasopressin. R, | OSMK
TGGAGTGCATGGAGT Ala AC002350 23543 tggagtgcatggagt 23529 12q24 Alu, IFNaR TGGAGTGCATGGAGT Ala AC002350 23543 tggagtgcatggagt 23529 12q24 Alu, IFNaR
プローブ Xaa ァクセシヨン番号 ヒットした位置 座位 blastx (expect=100)Probe Xaa accession number Hit position Locus blastx (expect = 100)
TGGAGTGCATGGAGT Ala AC004217 88822 tggagtgcatggagt 88808 12q24.1 ' Alu, HPK TGGAGTGCATGGAGT Ala AC004217 88822 tggagtgcatggagt 88808 12q24.1 'Alu, HPK
TTGGAGTTACTGGAGC Tyr AC002978 65893 tggagttactggagc 65907 12q24 clathrin LC, EP R(nonWS TTGGAGTTACTGGAGC Tyr AC002978 65893 tggagttactggagc 65907 12q24 clathrin LC, EP R (nonWS
TGGAGTTGTTGGAGT Cys AC000403 91715 tggagttgttggagt 91729 13 VHL, inhibin B TGGAGTTGTTGGAGT Cys AC000403 91715 tggagttgttggagt 91729 13 VHL, inhibin B
TGGAGCGGTTGGAGC Gly X97051 73621 tggagcggttggagc 73607 14q32.33 (IgD) polycystic kidney  TGGAGCGGTTGGAGC Gly X97051 73621 tggagcggttggagc 73607 14q32.33 (IgD) polycystic kidney
TGGAGTAGGTGGAGC Arg AC003024 15596 tggagtaggtggagc 15582 15q26 pksF  TGGAGTAGGTGGAGC Arg AC003024 15596 tggagtaggtggagc 15582 15q26 pksF
TGGAGTTTCTGGAGC Phe 93356 tggagtttctggagc 93370 16  TGGAGTTTCTGGAGC Phe 93356 tggagtttctggagc 93370 16
TGGAGTTCATGGAGT Ser U91318 102406 tggagttcatggagt 102392 16 ICAM1, MIBP1  TGGAGTTCATGGAGT Ser U91318 102406 tggagttcatggagt 102392 16 ICAM1, MIBP1
TGGAGTGTATGGAGT Val AC002289 10631 tggagtgtatggagt 10645 16 Alu  TGGAGTGTATGGAGT Val AC002289 10631 tggagtgtatggagt 10645 16 Alu
摩 、；: 摩;
TGGAGTTAATGGAGT AC002519 81768 tggagttaatggagt 81754 16 Rho, Notch TGGAGTTAATGGAGT AC002519 81768 tggagttaatggagt 81754 16 Rho, Notch
TGGAGCTGCTGGAGT Cys U91326 84127 tggagctgctggagt 84113 16 ll.2 NIPI-like, IL2ErinanHSiTGGAGCTGCTGGAGT Cys U91326 84127 tggagctgctggagt 84113 16 ll.2 NIPI-like, IL2ErinanHSi
Ί'Γ' (ί ΛΓ'ΤΓ' ΛΛΤΓ' Γ' ΛΓ'Τ AC()02:{():{ 10ί)Γ) t« , iiif : KiU : ίϊΟβ Hi 12 Ί'ΟΚ. OBR. iul nianyBR'Γ '(ί ΛΓ'ΤΓ' ΛΛΤΓ 'Γ' ΛΓ'Τ AC () 02: {(): {10ί) Γ) t «, iiif: KiU: ίϊΟβ Hi 12 Ί'ΟΚ. OBR. Iul niany
TGGAGCACTTGGAGC Thr AC002551 82245 tggagcacttgfagc 82259 16pl2.1 envelope, androgen R TGGAGTCCCTGGAGC Pro AC002299 162 tggag^ccctggagc 148 16pl2-pl3.1 CYCUN H, FN TGGAGCACTTGGAGC Thr AC002551 82245 tggagcacttgfagc 82259 16pl2.1 envelope, androgen R TGGAGTCCCTGGAGC Pro AC002299 162 tggag ^ ccctggagc 148 16pl2-pl3.1 CYCUN H, FN
：;；  : ;;
TGGAGTCACTGGAGT His U95737 16130 tggagtcactggagt 16144 16 l3.1 T Ra, HLAa  TGGAGTCACTGGAGT His U95737 16130 tggagtcactggagt 16144 16 l3.1 T Ra, HLAa
16374 tggagtcactggagt 16388 Notch, Pro-rich  16374 tggagtcactggagt 16388 Notch, Pro-rich
16599 tggagtcactggagt 16613 phosphatase, ORFB  16599 tggagtcactggagt 16613 phosphatase, ORFB
TGGAGCACTTGGAGC Thr AC004509 26031 tggagcacttggagc 26045 16p 13.3 TfcRb TGGAGCACTTGGAGC Thr AC004509 26031 tggagcacttggagc 26045 16p 13.3 TfcRb
TGGAGCCGTTGGAGC Arg AC004496 28217 tggagccgttggagc 28231 16pl3.3 mucin, ETl, TL12 fnonWS TGGAGCCGTTGGAGC Arg AC004496 28217 tggagccgttggagc 28231 16pl3.3 mucin, ETL, TL12 fnonWS
プロ一ブ Xaa ァクセシ 3ン番号 ヒットした位 座位 blastx (expect=100) Probe Xaa accession number Hit position Locus blastx (expect = 100)
TGGAGCCGCTGGAGC Arg AC004232 34550 tggagccgct gagc 34564 16 l3.3 , IgLk' AGPR  TGGAGCCGCTGGAGC Arg AC004232 34550 tggagccgct gagc 34564 16 l3.3, IgLk 'AGPR
TTGGAGTACTTGGAGC Thr AJ003147 151180 tggagtacttggagc 151166 16p l3.3 RanBP2  TTGGAGTACTTGGAGC Thr AJ003147 151180 tggagtacttggagc 151166 16p l3.3 RanBP2
TGGAGCGTGTGGAGC Val X71874 11520 tggagcgtgtggagc 11534 16q22.1 collagen a5IV  TGGAGCGTGTGGAGC Val X71874 11520 tggagcgtgtggagc 11534 16q22.1 collagen a5IV
TGGAGCAAATGGAGT Lys AC003663 114346 tggagcaaatggagt 114360 17 beta-D-glucosidase  TGGAGCAAATGGAGT Lys AC003663 114346 tggagcaaatggagt 114360 17 beta-D-glucosidase
TGGAGTCTCTGGAGC Leu AC003957 52898 tggagtctctggagc 52884 17 TIE-1, SEX, Rho,  TGGAGTCTCTGGAGC Leu AC003957 52898 tggagtctctggagc 52884 17 TIE-1, SEX, Rho,
TGGAGCAGATGGAGC Arg AC003971 76277 tggagcagatggagc 76263 18 LIM -1, TfcR  TGGAGCAGATGGAGC Arg AC003971 76277 tggagcagatggagc 76263 18 LIM -1, TfcR
TGGAGTGCATGGAGT Ala AD000812 30891 tggagtgcat ga^t 30905 19 Alu  TGGAGTGCATGGAGT Ala AD000812 30891 tggagtgcat ga ^ t 30905 19 Alu
TGGAGCTGCTGGAGT Cys AC004660 10008 tggagctgctggagt 10022 19 Repsl TGGAGCTGCTGGAGT Cys AC004660 10008 tggagctgctggagt 10022 19 Repsl
TGGAGCCCCTGGAGT Pro AC004490 14389 tggag;cccctggagt 14403 19 mucin, ataxin-2, N-WASPTGGAGCCCCTGGAGT Pro AC004490 14389 tggag; cccctggagt 14403 19 mucin, ataxin-2, N-WASP
TGGAGTCAGTiJdAdC ACA r.l 11 18:U Γ' t airt (； twe': 1 «：{<) 1 19 l2(NK TI'OR. I'RLR, OBR.-ric mTGGAGTCAGTiJdAdC ACA r.l 11 18: U Γ 't airt (; twe': 1 «: {<) 1 19 l2 (NK TI'OR. I'RLR, OBR.-ric m
TGGAGCAGATGGAGC Arg ACQ04QQ4 39010 treagcagatggagc 38996 19 l2 FRLR. TL12R. C7M- presumably a pseudogene CSFRb. ILllR(+st p codon) TGGAGCAGATGGAGC Arg ACQ04QQ4 39010 treagcagatggagc 38996 19 l2 FRLR.TL12R.C7M-presumably a pseudogene CSFRb.ILllR (+ st p codon)
39177 tggagcagatggagc 39163 TL3Ra(weak. 22 .nonWS) 39177 tggagcagatggagc 39163 TL3Ra (weak.22.nonWS)
TGGAGCACCTGGAGT Thr AD000685 21015 tggagcacctggagt 21001 19p l3.1 GM-GSFRbfa nWS+stQp)(TGGAGCACCTGGAGT Thr AD000685 21015 tggagcacctggagt 21001 19p l3.1 GM-GSFRbfa nWS + stQp)
TGGAGCTGATGGAGC *** AC002115 19ql3.1 Mpc2, Pro rich protein TGGAGCTGATGGAGC *** AC002115 19ql3.1 Mpc2, Pro rich protein
TGGAGCCAGTGGAGC Gin M63796 7622 tggagccagtggagc 7636 19ql3.3 NFCP, titin, Jagged 2  TGGAGCCAGTGGAGC Gin M63796 7622 tggagccagtggagc 7636 19ql3.3 NFCP, titin, Jagged 2
TGGAGTTACTGGAGT Tyr AC004505 31711 tggagttactggagt 31725 20 Gap junction  TGGAGTTACTGGAGT Tyr AC004505 31711 tggagttactggagt 31725 20 Gap junction
TGQAGTTGATGGAGC *** Z93016 31093 tggagttgatggagc 31079 20ql2-13.2 smaphoiin F, GHS-R, JAK2 TGQAGTTGATGGAGC *** Z93016 31093 tggagttgatggagc 31079 20ql2-13.2 smaphoiin F, GHS-R, JAK2
TGGAGTCAATGGAGT Gin (;77 579 tgga^tcaatggagrt 565 21(MX1) GLI, QU n,7Rf WS TGGAGTCAATGGAGT Gin (; 77 579 tgga ^ tcaatggagrt 565 21 (MX1) GLI, QU n, 7Rf WS
TGGAGTGCCTGGAGT Ala AF039907 29892 tggagtgcctssagt 29906 21 IgV, Cyt.Oxidase  TGGAGTGCCTGGAGT Ala AF039907 29892 tggagtgcctssagt 29906 21 IgV, Cyt.Oxidase
TGGAGTGTCTGGAGT Val AG000937 105 tggagtgtctggagt 91 21q peroxidasin TGGAGTGTCTGGAGT Val AG000937 105 tggagtgtctggagt 91 21q peroxidasin
ブロープ Xaa ァクセシヨン番号 ヒットした位置 座位 blastx (expect=100) Probe Xaa accession number Hit position Sitting blastx (expect = 100)
TGGAGTAAATGGAGT Lys AP000034 28803 tggagtaaatgigagt 28789 21qll.l * Na Ca exchanger  TGGAGTAAATGGAGT Lys AP000034 28803 tggagtaaatgigagt 28789 21qll.l * Na Ca exchanger
TTGGAGTAGGTGGAGT Arg AP000039 24900 tggagtaggtggagt 24914 21qll.l RNApol merase  TTGGAGTAGGTGGAGT Arg AP000039 24900 tggagtaggtggagt 24914 21qll.l RNApol merase
TGGAGTGAGTGGAGT Glu AP000035 21721 tggagtgagtggagt 21707 21qll.l smaphorin F  TGGAGTGAGTGGAGT Glu AP000035 21721 tggagtgagtggagt 21707 21qll.l smaphorin F
TGGAGTGTCTGGAGT Val AG000038 26164 tg agtgtctgsagt 26150 21qll.l Glycoprotein  TGGAGTGTCTGGAGT Val AG000038 26164 tg agtgtctgsagt 26150 21qll.l Glycoprotein
TGGAGTGCCTGGAGT Ala AP000045 7204 tggagtgcctggagt 7218 21qll.l i&v, TGGAGTGCCTGGAGT Ala AP000045 7204 tggagtgcctggagt 7218 21qll.l i & v,
TGGAGCATTTGGAGC He AP000052 93726 tggagcatttggagc 93740 21qll.l If H, TCF-3, CETP  TGGAGCATTTGGAGC He AP000052 93726 tggagcatttggagc 93740 21qll.l If H, TCF-3, CETP
TGGAGCCTCTGGAGC Leu AP000037 17581 tggagcctctggagc 17567 21qll.l Alu, BCGF  TGGAGCCTCTGGAGC Leu AP000037 17581 tggagcctctggagc 17567 21qll.l Alu, BCGF
TGGAGTGGGTGGAGT Gly AP000016 48480 tggagtggKtggag^ 48494 21q22.2 TPO  TGGAGTGGGTGGAGT Gly AP000016 48480 tggagtggKtggag ^ 48494 21q22.2 TPO
TGGAGTGAGTGGAGT Glu Z97055 151632 tggag^gagtggagt 151618 22 semaphoria H, CD44  TGGAGTGAGTGGAGT Glu Z97055 151632 tggag ^ gagtggagt 151618 22 semaphoria H, CD44
TGGAGCTGGTGGAGT Trp Z83856 8503 tggagctggtggagi 8489 22 ERF 05 TGGAGCTGGTGGAGT Trp Z83856 8503 tggagctggtggagi 8489 22 ERF 05
TGGAGTGGGTGGAGT Gly Z95113 69325 tfgagtgggtggagt 69311 22qll.2-qter factor H TGGAGTGGGTGGAGT Gly Z95113 69325 tfgagtgggtggagt 69311 22qll.2-qter factor H
TGGAGTGCATGGAGT Ala Z93784 36348 tggagtgcatggagt 36362 22qll.2-qter Alu, NF2  TGGAGTGCATGGAGT Ala Z93784 36348 tggagtgcatggagt 36362 22qll.2-qter Alu, NF2
TGGAGCCTCTGGAGT Leu AC002308 130741 tggagcctctggagt 130727 22qll.2 collagen al, Na channel  TGGAGCCTCTGGAGT Leu AC002308 130741 tggagcctctggagt 130727 22qll.2 collagen al, Na channel
TGGAGTCCCTGGAGC Pro AC000086 40705 tggagtccctggagc 40691 22qll.2 ADH, collagen  TGGAGTCCCTGGAGC Pro AC000086 40705 tggagtccctggagc 40691 22qll.2 ADH, collagen
TGGAGCATCTGGAGC He L77569 21088 tggagcatctggagc 21074 22qllDiGeorgeclathrin heavy chain 2  TGGAGCATCTGGAGC He L77569 21088 tggagcatctggagc 21074 22qllDiGeorgeclathrin heavy chain 2
2424a.¾gagcatctEgagc 24262 2424a.¾gagcatct Eg a g c 24262
TGGAGCAGCTGGAGC Ser AC000092 9817 tggagcagctggagc 9803 22qll.2 IgHv, PC binding  TGGAGCAGCTGGAGC Ser AC000092 9817 tggagcagctggagc 9803 22qll.2 IgHv, PC binding
TGGAGCAACTQGAGC Asn Z95116 64481 tggagcaactggagc 64495 22ql2.1 P150, TTt4RfWv NWSF*l TGGAGCAACTQGAGC Asn Z95116 64481 tggagcaactggagc 64495 22ql2.1 P150, TT t 4RfWv NWSF * l
TGGAGCTAGTGGAGC *** AC003071 114780 tggagctagtggagc 114794 22ql2.1-qter FQFRb TGGAGCTAGTGGAGC *** AC003071 114780 tggagctagtggagc 114794 22ql2.1-qter FQFRb
TGGAGCCCTTGGAGC Pro Z80902 2675 tggagcccttggagc 2661 collagen al TGGAGCCCTTGGAGC Pro Z80902 2675 tggagcccttggagc 2661 collagen al
TGGAGCTCTTGGAGT Ser Z79999 40825 tggagc cttgga^t 40839 22ql2-qter collagen al, TGGAGCTCTTGGAGT Ser Z79999 40825 tggagc cttgga ^ t 40839 22ql2-qter collagen al,
プ ブ Xaa ァクセシヨン番号 ヒヅ トした位 S 座位 blastx iexpect=100) (Pub Xaa accession number Hot position S locus blastx iexpect = 100)
TQGAGCCATTGGAGT His Z81308 12575 tgga^ccattg^agt 12561 22ql2-qter MYF-5, p53, I 4a  TQGAGCCATTGGAGT His Z81308 12575 tgga ^ ccattg ^ agt 12561 22ql2-qter MYF-5, p53, I 4a
Ι'Π: (； Λ(;(，（； Λ(ίΤ (； (； Λ(ΓΓ (51u Λ H1H(;:{7  Ι'Π: (; Λ (; (, (; Λ (ίΤ (; (; Λ (ΓΓ (51u Λ H1H (;: {7
TTGGAGTGAGTGGAGT Glu U62317 77740 tggagtgagtggagt 77726 22ql3 latropliilin-related  TTGGAGTGAGTGGAGT Glu U62317 77740 tggagtgagtggagt 77726 22ql3 latropliilin-related
TGGAGTGCATGGAGT Ala 31082 tggagtgcatggagt 31068 22ql3 Alu, SDSSS. AD7c-NTP  TGGAGTGCATGGAGT Ala 31082 tggagtgcatggagt 31068 22ql3 Alu, SDSSS. AD7c-NTP
TGGAGTTGTTGGAGT Cys AC002422 19151 tfgagttgttggagt 19137 X cGMP PDase  TGGAGTTGTTGGAGT Cys AC002422 19151 tfgagttgttggagt 19137 X cGMP PDase
TGGAGTGTCTGGAGT Val Z73418 31830 tggagt^tctggagt 31816 X W T-8D, Mi-2  TGGAGTGTCTGGAGT Val Z73418 31830 tggagt ^ tctggagt 31816 X W T-8D, Mi-2
TGGAGTCTTTGGAGT Leu Z83843 114972 tggagtctttggagt 114958 X reverse transcriptase  TGGAGTCTTTGGAGT Leu Z83843 114972 tggagtctttggagt 114958 X reverse transcriptase
TGGAGTCTCTGGAGT Leu Z99706 7749 tggagtctctggag 7735 X* Selenoprotein  TGGAGTCTCTGGAGT Leu Z99706 7749 tggagtctctggag 7735 X * Selenoprotein
TGGAGCAACTGGAGT Asn AC002420 70704 tggagcaactggagt 70690 X homeoproteinr OBRistop^ TGGAGCAACTGGAGT Asn AC002420 70704 tggagcaactggagt 70690 X homeoprotein r OBRistop ^
TGGAGCATGTGGAGT Met ■/772 1!)H 5702 tggagcatgtggagt 5688 X TcRb,DEIS3l  TGGAGCATGTGGAGT Met ■ / 772 1!) H 5702 tggagcatgtggagt 5688 X TcRb, DEIS3l
TGGAGTTCCTGGAGC Ser Z83131 4904 tggagttcctggagc 4890 X VPS41 homolog TGGAGTTCCTGGAGC Ser Z83131 4904 tggagttcctggagc 4890 X VPS41 homolog
1 1
TGGAGTGGCTGGAGC Gly AC004388 239975 tggagtggctggagc 239989 X GAP- mLTFRfs TGGAGTGGCTGGAGC Gly AC004388 239975 tggagtggctggagc 239989 X GAP-mLTFRfs
TGGAGTCTATGGAGC Leu Z70050 9934 tggagtctatggagc 9948 complement C8, C7 TGGAGTCTATGGAGC Leu Z70050 9934 tggagtctatggagc 9948 complement C8, C7
？  ?
TGGAGCTGTTGGAGC Cys L44140 112657 tggagctgttggagc 112671 rab GDI alpha, BDGF  TGGAGCTGTTGGAGC Cys L44140 112657 tggagctgttggagc 112671 rab GDI alpha, BDGF
TGGAGCTCATGGAGC Ser AC004383 144906 tggagctcatggagc 144892 RTase, transposon  TGGAGCTCATGGAGC Ser AC004383 144906 tggagctcatggagc 144892 RTase, transposon
TGGAGTAAATGGAGC Lys Z69732 31681 tggagtaaatggagc 31695 OT-R, acrosin  TGGAGTAAATGGAGC Lys Z69732 31681 tggagtaaatggagc 31695 OT-R, acrosin
TGGAGTTCGTGGAGC Ser Z92545 88703 tggagttcgtggagc 88717 PMK1  TGGAGTTCGTGGAGC Ser Z92545 88703 tggagttcgtggagc 88717 PMK1
TGGAGCTTCTGGAGC Phe AL008709 46089 tggagcttct gagc 46075 Xpll.23-Xpll.4r HC class la, HLA-C  TGGAGCTTCTGGAGC Phe AL008709 46089 tggagcttct gagc 46075 Xpll.23-Xpll.4r HC class la, HLA-C
TGGAGTTTCTGGAGT Phe U96409 116332 tggagtttctggaf 116346 Xp22 myosin H  TGGAGTTTCTGGAGT Phe U96409 116332 tggagtttctggaf 116346 Xp22 myosin H
TGQAGTTGCTGGAGT Cys 89544 tggagttgctggagt 89530 Xp22 QSIS  TGQAGTTGCTGGAGT Cys 89544 tggagttgctggagt 89530 Xp22 QSIS
X X X ¾ »..  X X X ¾ »..
1 1 11 1 1 11
Figure imgf000047_0001
Figure imgf000047_0001
TGGAGTTTCTGGAGT Phe AC002531 106698 tggag ttctggagt 106712 Y Alu, hpk  TGGAGTTTCTGGAGT Phe AC002531 106698 tggag ttctggagt 106712 Y Alu, hpk
TGGAGCAGTTGGAGC Ser AC004474 124745 tggagcagttggagc 124731 Y EGFR, Smad6 CO TGGAGCAGTTGGAGC Ser AC004474 124745 tggagcagttggagc 124731 Y EGFR, Smad6 CO
TGGAGTTTGTGGAGT Leu U26425 12899 tggagtttgtggagt 12913 PLCb2 P L Copposite^ TGGAGTTTGTGGAGT Leu U26425 12899 tggagtttgtggagt 12913 PLCb2 P L Copposite ^
TGGAGCAACTGGAGT Asn U96726 61672 tggagcaactggagt 61658 mouse DNA envelope mlLHRionnosite) TGGAGTCCCTGGAGC Pro QSEESSl 22244 tggagtccctggagc 22230 MHC class II CFTC lEni TGGAGCAACTGGAGT Asn U96726 61672 tggagcaactggagt 61658 mouse DNA envelope mlLHRionnosite) TGGAGTCCCTGGAGC Pro QSEESSl 22244 tggagtccctggagc 22230 MHC class II CFTC lEni
TGGAGCAGATGGAGC Arg AC002482 14276 tggagcagatggagc 14290 RG208O03 1-309, TcR, IL9RfnQn S) TGGAGCAGATGGAGC Arg AC002482 14276 tggagcagatggagc 14290 RG208O03 1-309, TcR, IL9RfnQn S)
TGGAGCTCTTGGAGC Ser U34879 24914 tggagctcttggagc 24928 EDH17B2 Large tegument protein TGGAGCTCTTGGAGC Ser U34879 24914 tggagctcttggagc 24928 EDH17B2 Large tegument protein
pnmmon foppsit. nonWSt pnmmon foppsit.nonWSt
TGGAGCCTTTGGAGC Leu Z15025 6359 tggagcctttggagc 6373 Bat2 bat2,mucin, TGGAGCCTTTGGAGC Leu Z15025 6359 tggagcctttggagc 6373 Bat2 bat2, mucin,
ΓτΜ-CSyRbfonnosite. stop) 過剰 （Redundant) クローンを影で示した。 白抜きとアンダーラインはそれそれヒットおよび偽ヒヅ ト （Pseudo-hits) を示す。 ΓτΜ-CSyRbfonnosite. Stop) Redundant clones are shaded. The outlines and underlines indicate hits and false hits, respectively.
さらにこの 28クローンのうち 4クローン （AC002303、 AC003112, AL008637及び AC004004) は複数個の既知へモポェチン受容体をヒットしたが AC004004は Trp- Ser-Xaa- Trp-Serモチーフの 3アミノ酸下流に終止コ ドンが存在していること から除外した。 残る 3クローンのうち AL008637は既知受容体である GM-CSF受容 体 5と考えられた。 AC002303は TI GRグループにより 1997年 6月 19日に登録された ヒト第 16染色体 16pl2領域由来の BACクローン CIT987-SKA-670B5であり全長 1315 30塩基対からなる （Lamerdin，J. E .， et al .， GenBank Report on AC003112, 1 997) o In addition, 4 of these 28 clones (AC002303, AC003112, AL008637 and AC004004) hit multiple known hemopoietin receptors, but AC004004 was a stop codon 3 amino acids downstream of the Trp-Ser-Xaa-Trp-Ser motif. Was excluded from the existence. Of the remaining three clones, AL008637 was considered to be GM-CSF receptor 5, a known receptor. AC002303 is a BAC clone CIT987-SKA-670B5 derived from the 16pl2 region of human chromosome 16, which was registered on June 19, 1997 by the TI GR group and has a total length of 1315 30 base pairs (Lamerdin, J. E., et al. ., GenBank Report on AC003112, 1 997) o
図 1に示す様に AC002303配列中唯一存在するプロ一ブ配列、 tggagtgaatggagt (40952-40966) を含む 40861- 41040の 180塩基を質問式 (query) とした BlastX 検索により TP0受容体、レブチン受容体を初めとする多数のへモポェチン受容体 が明らかな相同性を示したが質問式（query)配列と完全に一致する既知へモポ ェチン受容体はデータベースに登録されていなかった。また、上記 180塩基の配 列を中心に 5'、 3'両方向に順次 180塩基の配列を切り取って質問式 （query) と し、 上記条件にて BlastXスキャンを実施した結果さらに 2つの既知へモポェチ ン受容体と相同性を示す配列を 39181- 39360及び 4230卜 42480の領域に見出し同 一遺伝子の他のェクソンと考えられた （図 2 )。  As shown in Fig. 1, BlastX search using 180 bases of 40861-41040 including tggagtgaatggagt (40952-40966), which is the only probe sequence in the AC002303 sequence, A number of the first hemopoietin receptors showed obvious homology, but no known hemopoietin receptor that perfectly matched the query sequence was registered in the database. In addition, a 180-base sequence was sequentially cut in both 5 'and 3' directions centering on the 180-base sequence described above, which was used as a query, and a BlastX scan was performed under the above conditions. Sequences exhibiting homology to the receptor were found in the regions 39181-39360 and 4230-42480, and were considered to be other exons of the same gene (Fig. 2).
39181- 39360の部位には Pro- richモチーフ PAPPF、 42301-42480の部位には Box 1モチーフが保存されていた。 Trp-Ser-Xaa- Trp- Serモチーフを含むェクソンに 隣接する 3'側ェクソンは細胞膜貫通ドメインが載っているが、 このドメインは 他のへモポェチン受容体との相同性が低く BlastXスキャンでは検出されなかつ た。以上の結果より、上記 BACクローン C IT987- SKA- 670B5に新規へモポェチン受 容体遺伝子が存在する可能性が示唆された。  The Pro-rich motif PAPPF was conserved at 39181-39360, and the Box 1 motif was conserved at 42301-42480. The 3 'exon adjacent to the exon containing the Trp-Ser-Xaa- Trp- Ser motif has a transmembrane domain, but this domain has low homology to other hemopoietin receptors and is detected by BlastX scan. What. The above results suggested that the BAC clone C IT987-SKA-670B5 may have a novel hemopoietin receptor gene.
[実施例 2 ] RT- PCRによる NR8発現組織の検索  [Example 2] Search for NR8 expression tissues by RT-PCR
へモポェチン受容体の幾つかについては偽遺伝子の存在が報告されている（K ermouni ,A. et al . , Genomics 29 ( 2 ) , 1995 , 37卜 382、 Fukunaga, R. and Nag ata，S.， Eur. J. Biochem. , 1994, 220, 881-891)。 NR8が偽遺伝子でないことを 確認することおよび NR8発現組織の同定を目的に、 NR8遺伝子の転写産物の検索 を RT- PCR法にて実施した。 The presence of pseudogenes has been reported for some of the hemopoietin receptors (Kermouni, A. et al., Genomics 29 (2), 1995, 37, 382; Fukunaga, R. and Nag. ata, S., Eur. J. Biochem., 1994, 220, 881-891). To confirm that NR8 was not a pseudogene and to identify NR8-expressing tissues, NR8 gene transcripts were searched by RT-PCR.
上記 BACクローン AC002303の配列内において、既知サイ トカインレセプ夕一に アミノ酸翻訳レベルで、 広く保存されている複数のェクソン領域を予測し、 そ の予測したェクソン領域の配列上に以下のプライマ一を合成した （各プライマ 一の位置は図 5参照） 。  Within the sequence of the BAC clone AC002303, a plurality of widely-conserved exon regions were predicted at the amino acid translation level at a known sitekine receptor level, and the following primers were synthesized on the predicted exon region sequence. (See Figure 5 for the location of each primer.)
NR8-SN1； 5' - CCG GCT CCC CCT TTC AAC GTG ACT GTG ACC -3，（配列番号： 9 ) NR8-SN2 ; 5' - GGC AAG CTT CAG TAT GAG CTG CAG TAC AGG -3， （配列番号： 1 0 )  NR8-SN1; 5'-CCG GCT CCC CCT TTC AAC GTG ACT GTG ACC-3, (SEQ ID NO: 9) NR8-SN2; 5'-GGC AAG CTT CAG TAT GAG CTG CAG TAC AGG-3, (SEQ ID NO: Ten )
NR8-AS1； 5' - ACC CTC TGA CTG GGT CTG AAA GAT GAC CGG -3， （配列番号： 1 1 )  NR8-AS1; 5'-ACC CTC TGA CTG GGT CTG AAA GAT GAC CGG-3, (SEQ ID NO: 11)
NR8-AS2; 5' - CAT GGG CCC TGC CCG CAC CTG CAG CTC ATA -3， （配列番号： 1 2 )  NR8-AS2; 5'-CAT GGG CCC TGC CCG CAC CTG CAG CTC ATA-3, (SEQ ID NO: 12)
Human Fetal Multiple Tissuue cDNA Panel (Clontech#K1425- 1 )を銪型とし て用いて、 上記プライマーの組合わせによる RT-PCRを試みた。 PCRには Advanta ge cDNA Polymerase Mix (Clontech#8417-l )を用い、 Perkin Elmer Gene Amp PCR System 2400サ一マルサイクラ一を使用し、下記の実験条件にて実施した。 即ち、 PCR条件は、 94°Cで 4分、 「94°Cで 20秒、 72°Cで 3分」 を 5サイクル、 「9 4°Cで 20秒、 70°Cで 3分」 を 5サイクル、 「94°Cで 20秒、 68°Cで 3分」 を 28サイク ル、 72°Cで 4分、 および 4°Cにて終結、 である。  Using the Human Fetal Multiple Tissuue cDNA Panel (Clontech # K1425-1) as type III, RT-PCR was performed using the combination of the above primers. The PCR was performed using Advantage cDNA Polymerase Mix (Clontech # 8417-l) and a Perkin Elmer Gene Amp PCR System 2400 thermal cycler under the following experimental conditions. That is, PCR conditions were 4 cycles at 94 ° C, 5 cycles of `` 20 seconds at 94 ° C, 3 minutes at 72 ° C '', and 5 cycles of `` 20 seconds at 94 ° C, 3 minutes at 70 ° C ''. The cycle, "20 seconds at 94 ° C, 3 minutes at 68 ° C", is 28 cycles, 4 minutes at 72 ° C, and ends at 4 ° C.
図 5に示したプライマーの位置から、 SN1/ASK SN1/AS2, SN2/ASU SN2/AS2 の組み合わせでそれそれ、 330bp、 258bp、 234bp、 162bpのサイズのバンドの増 幅が期待される。 ヒト胎児肝、 脳、 骨格筋 c D N Aを铸型として検討した結果、 胎児肝についてのみそれそれのプライマーの組み合わせで予想されたサイズの 明瞭なバンドが観察された （図 3 ) 。 これに対して胎児脳 c D N Aでは全く増幅を認めず、 胎児骨格筋では約 650b Pのバンドと 400-500bpのブロードなバンドが観察された。 しかし胎児骨格筋の 場合のバンドのサイズは異なる組み合わせのプライマーを用いた場合でも一定 であったことから何らかの理由による非特異的な増幅と考えられた。 From the positions of the primers shown in FIG. 5, it is expected that bands of 330 bp, 258 bp, 234 bp, and 162 bp in size will be amplified by the combination of SN1 / ASK SN1 / AS2, SN2 / ASU SN2 / AS2, respectively. As a result of examining human fetal liver, brain and skeletal muscle cDNA as type III, a clear band of the expected size was observed only for fetal liver with each primer combination (Fig. 3). In contrast, no amplification was observed in fetal brain cDNA, and a broad band of about 650 bp and a broad band of 400-500 bp were observed in fetal skeletal muscle. However, in the case of fetal skeletal muscle, the band size was constant even when different combinations of primers were used, so this was considered to be nonspecific amplification for some reason.
得られた PCR産物を、 pGEM-T Easy vector (Promega#A1360)にサブクロ一ニン グし、 塩基配列を決定した。 PCR産物の pGEM- T Easy vectorへの組換えは、 T4 DNA Ligase(Promega#A1360 )によって、 4°C/12時間の反応をおこなった。 PCR産 物と pGEM-T Easy vectorの遺伝子組換え体は、 大腸菌株 DH5a ( Toyobo#DNA-90 3 )を形質転換することによって得られた。  The obtained PCR product was subcloned into pGEM-T Easy vector (Promega # A1360), and the nucleotide sequence was determined. The recombination of the PCR product into the pGEM-T Easy vector was performed at 4 ° C. for 12 hours using T4 DNA Ligase (Promega # A1360). Transgenic products of the PCR product and the pGEM-T Easy vector were obtained by transforming E. coli strain DH5a (Toyobo # DNA-903).
また、 遺伝子組換え体の選別には、 Insert Check Ready ( Toyobo#PIK-101 ) を用いた。 さらに、 塩基配列の決定には、 dRhodamine Terminator Cycle Sequ encing Kit (ABI/Perkin Elmer#4303141 )を使用し、 ABI PRISM 377 DNA Seque ncer によって解析をおこなった。独立する 10クローンの遺伝子組換え体に対し、 全ィンサート断片の塩基配列を決定した結果、 全クローンともに単一の塩基配 列を示した。 この得られた配列が、 NR8の部分塩基配列であることを確認した。  In addition, Insert Check Ready (Toyobo # PIK-101) was used for selecting the recombinants. Further, the nucleotide sequence was determined using ABI PRISM 377 DNA Sequencer using dRhodamine Terminator Cycle Sequencing Kit (ABI / Perkin Elmer # 4303141). As a result of determining the nucleotide sequence of all the insert fragments for 10 independent clones of the recombinant, all the clones showed a single nucleotide sequence. It was confirmed that the obtained sequence was a partial nucleotide sequence of NR8.
[実施例 3 ] 5' -及び 3， -RACE法による完全長 c D N Aクロ一ニング  [Embodiment 3] Full-length cDNA closing by 5'- and 3, -RACE method
次にこの胎児肝由来 c D N Aを用いて完全長 c D N Aを得るべく、 5，-RACE、 3' -RACE法を実施した （図 4 ) 。  Next, 5, -RACE and 3'-RACE methods were performed to obtain full-length cDNA using the fDNA derived from fetal liver (FIG. 4).
3-1 ) 5， -RACE法  3-1) 5, -RACE method
前記の NR8- AS1プライマ一を一次 PCRに用い、 また、 NR8- AS2プライマ一を二次 PCRに用いて 5' RACE- PCRを試みた。 錶型として Human Fetal Liver Marathon-Re ady cDNA Library (Clontech#7403- 1 )を用い、 PCR実験には Advantage cDNA Po lymerase Mixを使用した。Perkin Elmer Gene Amp PCR System 2400サーマルサ ィクラ一を使用し、下記の PCR条件で行った結果、選択的スプライシングによる 二種類のサイズを示す PCR産物が得られた。  5 ′ RACE-PCR was attempted using the NR8-AS1 primer for primary PCR and the NR8-AS2 primer for secondary PCR. Human Fetal Liver Marathon-Ready cDNA Library (Clontech # 7403-1) was used as type 錶, and Advantage cDNA Polymerase Mix was used for PCR experiments. As a result of using the Perkin Elmer Gene Amp PCR System 2400 thermal cycler under the following PCR conditions, two sizes of PCR products were obtained by alternative splicing.
一次 PCRの条件は、 94°Cで 4分、 「94°Cで 20秒、 72°Cで 4分」 を 5サイクル、 「9 4°Cで 20秒、 70°Cで 4分」 を 5サイクル、 「94°Cで 20秒、 68°Cで 4分」 を 28サイク ル、 72°Cで 4分、 および 4°Cにて終結である。 The primary PCR conditions were 94 ° C for 4 minutes, 5 cycles of `` 20 seconds at 94 ° C, 4 minutes at 72 ° C '', `` 9 5 cycles of `` 20 seconds at 4 ° C, 4 minutes at 70 ° C '', 28 cycles of `` 20 seconds at 94 ° C, 4 minutes at 68 ° C '', 4 minutes at 72 ° C, and 4 ° C Is over.
二次 PCRの条件は、 94°Cで 4分、 「94°Cで 20秒、 70°Cで 3分 30秒」 を 5サイクル、 「94°Cで 20秒、 68°Cで 3分 30秒」 を 28サイクル、 72°Cで 4分、 および 4°Cにて終結 である。  The conditions for the secondary PCR were as follows: 4 cycles at 94 ° C, 5 cycles of `` 20 seconds at 94 ° C, 3 minutes and 30 seconds at 70 ° C '', `` 20 seconds at 94 ° C, 3 minutes at 68 ° C 30 Seconds ”for 28 cycles, 4 minutes at 72 ° C, and termination at 4 ° C.
得られた二種類の PCR産物は双方とも、 前記同様、 pGEM-T Easy vector にサ ブクローニングし、 独立する 16クローンの遺伝子組換え体に対し、 全インサー ト断片の塩基配列を決定した。 塩基配列の決定には前記同様、 dRhodamine Ter minator Cycle Sequencing Kitを使用し、 ABI PRISM 377 DNA Sequencerによつ て解析をおこなった。 その結果、 塩基対の長さ、 及び配列の相違により、 14ク ローンと 2クローンの二種類のグループに区別することができた （これは、 後 述するが、 選択的スプライシングに起因する産物の相違であり、 独立する 14ク ローンのグループはゲノム配列においてェクソン 5に相当する配列を含み、残る 独立した 2クローンのグループにはこの配列が含まれていない） 。  In the same manner as described above, both of the obtained two types of PCR products were subcloned into the pGEM-T Easy vector, and the nucleotide sequences of all the insert fragments were determined for 16 independent gene recombinants. The nucleotide sequence was determined using the dRhodamine Terminator Cycle Sequencing Kit and analyzed using the ABI PRISM 377 DNA Sequencer as described above. As a result, it was possible to distinguish between 14 clones and 2 clones based on the differences in base pair length and sequence (this will be described later. The difference is that a group of 14 independent clones contains a sequence corresponding to exon 5 in the genomic sequence, and a group of two independent clones does not include this sequence.)
3-2) 3，- RACE法  3-2) 3,-RACE method
前記の NR8-SN1プライマーを一次 PCRに用い、 また、 NR8- SN2プライマ一を二次 PCRに用いて 3' RACE-PCRを試みた。 鎵型として 5' RACE- PCR同様、 Human Fetal L iver Marathon-Ready cDNA Libraryを用い、 PCR実験には Advantage cDNA Poly merase Mixを使用した。 PCR条件は前記、 3-1 )に示した通りで行った結果、 シン グルバンドの PCR産物が得られた。  3 ′ RACE-PCR was attempted using the NR8-SN1 primer for primary PCR and the NR8-SN2 primer for secondary PCR. As in the case of 5 ′ RACE-PCR, Human Fetal Liver Marathon-Ready cDNA Library was used as type 鎵, and Advantage cDNA Polymerase Mix was used for PCR experiments. As a result of performing PCR under the conditions described in 3-1) above, a single-band PCR product was obtained.
得られた PCR産物を、前記同様、 pGEM- T Easy vectorにサブクロ一ニングし、 独立する 12クローンの遺伝子組換え体に対し、 全ィンサート断片の塩基配列を 決定した。 塩基配列の決定は、 前記同様、 dRhodamine Terminator Cycle Sequ encing Kitを使用し、 ABI PRISM 377 DNA Sequencerによって解析をおこなった。 この結果、 独立した全 12クローンともに単一の塩基配列を示した。  The obtained PCR product was subcloned into the pGEM-T Easy vector in the same manner as described above, and the nucleotide sequence of all the insert fragments was determined for 12 independent clones of the gene recombinant. The nucleotide sequence was determined by the ABI PRISM 377 DNA Sequencer using the dRhodamine Terminator Cycle Sequencing Kit as described above. As a result, all 12 independent clones showed a single nucleotide sequence.
5' -RACE, 3， -RACEでそれそれ増幅された断片 （約 l . lkbと 1.2kb) の塩基配列 を解析した結果、 それそれの断片は互いに約 260bp重複して 5'側、 3'側に伸びた 配列で NR8 mMAのほぼ全長を含むと考えられたことからこれを連結して全長 c D N A (NR8ひ） とした （図 5、 図 6 ) 。 なお、 NR8ひ c D N A (配列番号： 2 ) を含有するプラスミ ドは pGEM- NR8ひと命名され、 該プラスミ ドを含有する大腸 菌は、 平成 10年 （1998年） 10月 9日に工業技術院生命工学工業技術研究所（茨城 県つくば市東 1丁目 1番 3号） に寄託番号 FERM BP- 6543として、 ブダペスト条 約に基づき国際寄託されている。 Base sequence of 5'-RACE, 3, and -RACE amplified fragments (about l.lkb and 1.2kb) As a result of the analysis, it was considered that the respective fragments overlapped by about 260 bp and extended to the 5 ′ side and the 3 ′ side, and were considered to contain almost the entire length of NR8 mMA. NR8) (Fig. 5, Fig. 6). The plasmid containing the NR8 cDNA (SEQ ID NO: 2) was named pGEM-NR8, and the E. coli containing the plasmid was reported on October 9, 1998 by the Institute of Industrial Science and Technology. It has been deposited internationally under the Budapest Convention at the Biotechnology Research Institute (1-3 1-3 Higashi, Tsukuba, Ibaraki Prefecture) under the deposit number FERM BP-6543.
図 5および図 6に示す様に、 NR8ひ c D N Aの 0RFは塩基番号 441から始まる M etが 39bp上流のインフレーム （in frame) 終止コ ドンの存在から閧始コ ドンと 考えられ、塩基番号 1524から始まる 2個の終止コドンで終了する。 N末端側から 順に、 典型的な分泌シグナル配列、 他のへモポェチン受容体メンバーに保存さ れた Cys残基を含むリガンド結合部位と考えられるドメイン、 Pro-リツチモチー フ、 Trp-Ser-Xaa-Trp-Serモチーフ、 細胞膜貫通ドメイン、 シグナル伝達に関与 すると考えられる Boxlモチーフ等のへモポェチン受容体の特徴を備えている。 以上の結果から、 NR8遺伝子は新規なへモポェチン受容体をコ一ドしているもの と考えられた。  As shown in FIG. 5 and FIG. 6, the 0RF of NR8 cDNA is considered to be the start codon based on the presence of an in-frame (39 frame) upstream in-frame stop codon starting from base number 441. Ends with two stop codons starting at 1524. From the N-terminal side, a typical secretory signal sequence, a domain considered to be a ligand binding site containing a Cys residue conserved in other hemopoietin receptor members, Pro-ritzimochi, Trp-Ser-Xaa-Trp It has features of hemopoietin receptor such as -Ser motif, transmembrane domain, Boxl motif thought to be involved in signal transduction. These results suggest that the NR8 gene encodes a novel hemopoietin receptor.
RACE法で増幅された断片を解析した結果、 スプライス変異体の存在が示唆さ れた。塩基配列解析の結果、 この変異体は上記 NR8ひの Pro-リツチモチーフを含 む約 150bpを欠失していることが明らかとなった。 さらに AC002303の配列と NR8 ひとの比較からェクソン ·ィントロンの類推を行った結果 （表 9 ) 、 上記変異 体は表 9中の第 5ェクソンを選択的スプライシングにより欠失しているものと 考えられた。 エクリン # inAC002303 # in NR8 特徴 Analysis of the fragments amplified by the RACE method indicated the presence of splice variants. As a result of nucleotide sequence analysis, it was found that this mutant lacked about 150 bp containing the Pro-ritichi motif of NR8. Furthermore, as a result of analogy of exon intron from the comparison of the sequence of AC002303 and NR8 human (Table 9), it was considered that the above mutant lacks the fifth exon in Table 9 by alternative splicing. . Ecrine # inAC002303 # in NR8 Features
1 <1 1-424 インフレ-ム終始]ドン  1 <1 1-424 Inflation Full] Don
2 26334-26398 425-489 閧始コにン， シク ΛΛ フ°チト *  2 26334-26398 425-489
3 30625-30727 490-592 保存 Cys残基  3 30625-30727 490-592 Conserved Cys residue
4 33766-33965 593-792 保存 Cys 残基， Ν· リ:！シレ-シヨン部位  4 33766-33965 593-792 Conserved Cys residue, Shire part
5 39240-39394 793-947 Pro-リッチ -フ (PAPPF), N-ク,リコシレ-シヨン部位 5 39240-39394 793-947 Pro-Rich-P (PAPPF), N-C , Ricoh-Resistance
6 40820-40997 948-1125 gtWSEWSdp t -フ  6 40820-40997 948-1125 gtWSEWSdp t -f
7 41455-41554 1126-1225 膜貫通に ン CD 7 41455-41554 1126-1225 Transmembrane CD
8 42285-42366 1226-1307 Boxl (IWAVPSP) 8 42285-42366 1226-1307 Boxl (IWAVPSP)
9a 44812-44909 1308-1405* エクリン 10への接続, Box2様配列 （PSTLEVYSCH), 非典型的な I /イント Uン境界  9a 44812-44909 1308-1405 * Connection to eccrine 10, Box2-like sequence (PSTLEVYSCH), atypical I / Int boundary
9b 44812-45922< 1308-2465** 二重の終始コドン, Box2様配列 (PSTLEVYSCH, PAELVESDG), poly A  9b 44812-45922 <1308-2465 ** Double stop codon, Box2-like sequence (PSTLEVYSCH, PAELVESDG), poly A
10 45441-45922< 1406-1934* 二重の終始コドン, poly A  10 45441-45922 <1406-1934 * Double stop codon, poly A
NR8 * ： Iクリン l+2+3+4+5+6+7+8+9a+10  NR8 *: I clean l + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9a + 10
NR8 J3 ： Iクリン l+2+3+4+6+7+8+9a+10 (可溶型および膜貫通 (-signal)型のための 2つのオルタナティ： T読み枠）  NR8 J3: I-crine l + 2 + 3 + 4 + 6 + 7 + 8 + 9a + 10 (two alternatives for soluble and transmembrane (-signal): T reading frame)
NR8 ァ "： エクリン l+2+3+4+5+6+7+8+9b NR8a ": eccrine l + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9b
この変異体（NR8 ?)は第 4ェクソンに第 6ェクソンが直結してフレームシフ 卜が起こり トランケ一ティ ドフォーム（runcated form)の可溶性受容体をコ一 ドすることが可能である。 またェクソンとイントロンの境界は殆どの場合コン センサス配列をとつているが、 唯一第 9ェクソン （Exon 9a) と第 9イン卜ロン の境界だけ acc/acggagとコンセンサス配列（nag/gtgagt等） とは異なっている。 なお、 NR8 ? cDNA (配列番号： 4) を含有するプラスミ ドは pGEM- NR85と命 名され、 該プラスミ ドを含有する大腸菌は、 平成 10年 （1998年） 10月 9日に工業 技術院生命工学工業技術研究所 （茨城県つくば巿東 1丁目 1番 3号） に寄託番 号 FEM BP- 6544として、 ブダぺスト条約に基づき国際寄託されている。 In this mutant (NR8?), The sixth exon is directly linked to the fourth exon, and a frame shift occurs to encode a soluble receptor in a truncated form. In most cases, the boundary between exon and intron has a consensus sequence, but only the boundary between ninth exon (Exon 9a) and ninth intron is acc / acggag and consensus sequence (nag / gtgagt, etc.). Is different. The plasmid containing the NR8 cDNA (SEQ ID NO: 4) was named pGEM-NR85, and E. coli containing the plasmid was found on October 9, 1998 by It has been deposited internationally under the Budapest Treaty under the deposit number FEM BP-6544 at the Engineering Research Institute of Technology (1-3, Tsukuba East, Ibaraki Prefecture).
[実施例 4 ] ノ一ザンブロッテイング  [Example 4] Northern blotting
各ヒト臓器、 及びヒト癌細胞株における NR8の遺伝子発現分布、 及び、 遺伝子 発現様態を解析するために、実施例 3にて得られた全ての cDN A断片を基に調 製した NR8ひ蛋白質全長をコ一ドする cDN Aをプローブとして用い、ノーザン 解析を行った。 プローブの調製は、 Mega Prime Kit(Amersham，cat#RPN1607)を 使用し、 [ひ-32 P]dCTP (Amersham，cat#AA0005)によって標識した。 In order to analyze the gene expression distribution of NR8 in each human organ and human cancer cell line, and the gene expression mode, the full-length NR8 protein prepared based on all the cDNA fragments obtained in Example 3 Using the cDNA as a probe, Northern analysis was performed. Preparation of probe, using the Mega Prime Kit (Amersham, cat # RPN1607), [ shed - 32 P] labeled by dCTP (Amersham, cat # AA0005) .
ノーザンブロッ トは Human Multiple Tissue Northern(MTN) Blot (Clontech #7760-1), Human MTN Blot IV (Clontech#7766-l ), Human Cancer Cell Line MTN Blot (Clontech#7757-l) を用いた。 ハイブリダィゼーシヨンは Express Hyb Hybridization Solution (Clontech#8015- 2)を使用した。  The Northern blot used Human Multiple Tissue Northern (MTN) Blot (Clontech # 7760-1), Human MTN Blot IV (Clontech # 7766-l), and Human Cancer Cell Line MTN Blot (Clontech # 7757-l). Hybridization used Express Hyb Hybridization Solution (Clontech # 8015-2).
ハイブリダイゼーションの条件は 68°C/30分のプレハイブリダイゼ一シヨン の後、 68°C/14時間のハイプリダイゼ一シヨンを実施した。下記の条件にて洗浄 をおこなった後、 Imaging Plate (FUJI#BAS-III)に露光し、 Image Analyzer (F UJIX, BAS-2000 II)によって、 NR8 mRNAの遺伝子発現を検出した。洗浄条件は、 (1) lx SSC I 0.1% SDS,室温で 5分、 (2) lx SSC / 0.1% SDS，50°Cで 30分、 (3) O.lx SSC I 0.1¾ SDS, 50。Cで 30分である。  Hybridization conditions were 68 ° C / 30 minutes of prehybridization followed by 68 ° C / 14 hours of hybridization. After washing under the following conditions, the plate was exposed to an imaging plate (FUJI # BAS-III), and the gene expression of NR8 mRNA was detected by an Image Analyzer (FUJIX, BAS-2000 II). The washing conditions were (1) lx SSC I 0.1% SDS, 5 minutes at room temperature, (2) lx SSC / 0.1% SDS, 30 minutes at 50 ° C, (3) O.lx SSC I 0.1¾ SDS, 50. 30 minutes in C.
NR8の各臓器における発現をノーザンプロッティングにより解析した結果を 図 12に示す。 5kbおよび 3-4kbに 2本の計 3本の異なるサイズの mRN Aが成人 肺、 脾臓、 胸腺、 骨格筋、 膝臓、 小腸、 末梢白血球、 子宮に検出された。 さら に造血系細胞株を含む種々の細胞株についても同様に検討したところ、 前骨髄 性白血病細胞株である HL60とバーキヅトリンフォーマ由来 Rajiの 2株において 同じサイズのバンドを認めた。 Analysis of NR8 expression in each organ by Northern plotting See Figure 12. Three different sizes of mRNA, two at 5 kb and 3-4 kb, were detected in adult lung, spleen, thymus, skeletal muscle, knee, small intestine, peripheral leukocytes, and uterus. Furthermore, various cell lines, including hematopoietic cell lines, were examined in the same manner. Bands of the same size were observed in the HL60 promyelocytic leukemia cell line and the Raji-derived Raji-derived Raji line.
[実施例 5] プラークスクリーニング  [Example 5] Plaque screening
ノーザン解析による NR8遺伝子の発現解析の結果、 NR8遺伝子の発現が認めら れた各ヒト臓器、 および各ヒト癌細胞株のそれそれにおいて、 少なくともサイ ズの異なる 3種類の特異的な mRN Aのバンドが検出された。 しかしながら、本 発明者らは上記実施例においては、 2種類の選択的スプライシング変異体、即ち NR8ひおよび の遺伝子の単離にしか成功していない。そこで、第 3の選択的 スプライシング変異体の遺伝子単離を目的としたプラークスクリーニングを試 みた。 cDNAライブラリーとして、 前述のノーザン解析の結果、 強い NR8遺伝 子の発現が認められた Human Lymph Node (Clontech,cat#HL5000a) を用いた。 また、 プローブには NR8ひ c DN A断片を用いた。 プローブの標識は、 Mega Pr ime Kit (Amersham,cat#RPN1607) を使用し、 [ひ-32 P]dCTP (Amersham,cat#AAO 005) によってラジオアイソトープ標識した。 約 7.2 xlO5プラークの Human Lym ph Node cDNA Libraryを、 Hybond N ( + ) (Amersham,cat#RPN303B)付電荷ナイ口 ン膜にブロッテイングし、 一次スクリーニングを行った。 ハイブリダィゼ一シ ヨンは、 Rapid Hybridization Buffer (Amersham,cat#RPN1636) を使用した。 ハイプリダイゼーシヨンの条件は、 65°Cで 1時間のプレハイプリダイゼーシヨン の後、 65°Cで 14時間のハイブリダィゼーシヨンを実施した。 （l)lxSSC/0.1 SDS，室温で 15分、 (2) l SSC/0.1¾ SDS,58°Cで 30分、 (3) O.l SSC/0.1% SD S,58°Cで 30分の条件にて洗浄を行った後、 X線フィルム （Kodak, cat#165-1512) に露光し、 NR8陽性プラークを検出した。 As a result of Northern blot analysis of NR8 gene expression, at least three types of specific mRNA bands with at least different sizes in each human organ in which NR8 gene expression was observed and in each human cancer cell line Was detected. However, the present inventors have only succeeded in isolating the genes of two alternative splicing variants, namely NR8 and NR8, in the above example. Therefore, a plaque screening aimed at isolating the gene for the third alternative splicing mutant was attempted. As a cDNA library, a Human Lymph Node (Clontech, cat # HL5000a) in which strong NR8 gene expression was confirmed as a result of the Northern analysis described above was used. In addition, an NR8 cDNA fragment was used as a probe. Labeled probes, Mega Pr ime Kit (Amersham, cat # RPN1607) was used to [shed - 32 P] dCTP (Amersham, cat # AAO 005) were radioisotope-labeled by. The Human Lym ph Node cDNA Library about 7.2 XLO 5 plaques were blotted to Hybond N (+) (Amersham, cat # RPN303B) with charge Nai port down films were primary screening. For the hybridization, Rapid Hybridization Buffer (Amersham, cat # RPN1636) was used. The conditions of the hybridization were pre-hybridization at 65 ° C for 1 hour, and then hybridization at 65 ° C for 14 hours. (L) lxSSC / 0.1 SDS, 15 minutes at room temperature, (2) l SSC / 0.1¾ SDS, 30 minutes at 58 ° C, (3) Ol SSC / 0.1% SDS, 30 minutes at 58 ° C After washing, the plate was exposed to X-ray film (Kodak, cat # 165-1512) to detect NR8 positive plaque.
その結果、 陽性あるいは疑陽性を示す、 独立した 16クローンが得られた。 一 次スクリーニングにより得られたこの 16クローンに対し、 同様に二次スクリ一 ニングを行った結果、 NR8陽性を示す、独立した 15クローンのプラークの単離に 成功した。 これら 15クローンのインサート断片は、 人 gtlOベクタ一クローニン グ部位の両端に位置するプライマ一対によって PCR増幅された。 PCR反応には、 A dvantage cDNA polymerase Mix (Clontech#8417 - 1) を用い、 Perkin Elmer Ge ne Amp PCR System 2400サ一マルサイクラ一を使用し、 下記の実験条件にて実 施した。 即ち、 94°Cで 4分、 「94°Cで 20秒、 70°Cで 4分」 を 5サイクル、 「94°Cで 20秒、 68°Cで 4分」 を 30サイクル、 72°Cで 4分、 および 4°Cにて終結である。 As a result, 16 independent clones showing positive or false positive were obtained. one Secondary screening was similarly performed on these 16 clones obtained by the secondary screening, and as a result, plaques of 15 independent clones showing NR8 positivity were successfully isolated. The insert fragments of these 15 clones were amplified by PCR using a pair of primers located at both ends of the human gtlO vector-cloning site. The PCR reaction was carried out using Advantage cDNA polymerase Mix (Clontech # 8417-1) and Perkin Elmer Gene Amp PCR System 2400 Thermal Cycler under the following experimental conditions. 4 cycles at 94 ° C, 5 cycles of “20 seconds at 94 ° C, 4 minutes at 70 ° C”, 30 cycles of “20 seconds at 94 ° C, 4 minutes at 68 ° C”, 72 ° C For 4 minutes and at 4 ° C.
得られた PCR産物は、上記と同様に、 pGEM- T Easy vectorにサブクロ一ニング し、 インサート断片の塩基配列を決定した。 塩基配列の決定には、 BigDye Ter minator Cycle Sequencing SF Ready Reaction Kit (AB I/Perkin Elmer#43031 50) を使用し、 ABI PRISM 377 DNA Sequencerによって解析を行った。 その結果、 得られた 15クローンのうち、少なくとも 2クローンにおいて、 NR8ひの C末端近傍 に 177アミノ酸の挿入を認め、この部分が NR8遺伝子の第 9ィントロンに由来し、 NR8ひではスプライシングにより除去されていることから、 この第 3の選択的ス プライシング変異体を、 本発明者らは NR8ァと命名した。 なお、 NR8ァ c D N A (配列番号： 8 ) を含有するプラスミ ドは PGEM-NR8ァと命名され、 該プラスミ ドを含有する大腸菌は、 平成 10年（1998年） 10月 9日に工業技術院生命工学工業 技術研究所 （茨城県つくば巿東 1丁目 1番 3号） に寄託番号 FERM BP-6545とし て、 ブダペスト条約に基づき国際寄託されている。  The obtained PCR product was subcloned into the pGEM-T Easy vector in the same manner as described above, and the nucleotide sequence of the insert fragment was determined. The nucleotide sequence was determined using the ABI PRISM 377 DNA Sequencer using the BigDye Terminator Cycle Sequencing SF Ready Reaction Kit (ABI / Perkin Elmer # 4303150). As a result, in at least two of the 15 clones obtained, an insertion of 177 amino acids was observed near the C-terminus of NR8, which originated from the 9th intron of the NR8 gene and was removed by splicing in NR8. Therefore, we named this third alternative splicing variant NR8a. The plasmid containing the NR8 cDNA (SEQ ID NO: 8) was named PGEM-NR8, and E. coli containing the plasmid was identified on October 9, 1998 by the Institute of Industrial Science and Technology. It has been deposited internationally under the Budapest Treaty under the deposit number FERM BP-6545 at the Research Institute for Biotechnology Industry (Tsukuba-Higashi 1-3-1, Ibaraki Prefecture).
ここで得られた 1 5クローンの内、 その後さらに上記 2クロ一ン以外にも 4 クローンを選択して同様に塩基配列を解析した。 その結果、 選択された合計 6 クローンの内、 2クローンが NR8 ?の塩基配列を示し、残る 4クローンは全て N R8ァの塩基配列を示した。 従ってここで塩基配列を解析した 6クローンの中に NR8 ひの配列は含まれていなかった。また、配列を決定した NR8ァ cDNAクロ一 ンの 3'-UTOは、 3'-RACEによって得られた NR8ひの 3'- UTR末端配列（3UTR-1 )よ り 483 bp 伸長された部位に poly A テールが付加（3UTR-2 )される cDNAクロ一 ンと、 2397 bp伸長された部位に poly A テールが付加（3UTR-3 )される cDNAク ローンの存在が確認された。 一方、 上記によって配列を決定した 2クローンの においては、 何れも 3UTR- 3の塩基配列を含んでいた。 下記の表 1 0に、 これまでに得られた cDNAクローンが保持していた 3' 末端非翻訳領域配列をま とめた。また、 NR8ァ cDNA配列の翻訳終止コドンから起算した場合の 3UTR- 1、 3UTR-2及び 3UTR- 3の塩基配列を、 それそれ配列番号： 2 3、 配列番号： 2 4 及び配列番号： 2 5に示した。 Of the 15 clones obtained here, 4 clones were further selected in addition to the above-mentioned 2 clones, and the nucleotide sequence was similarly analyzed. As a result, out of a total of six selected clones, two clones showed the nucleotide sequence of NR8 ?, and the remaining four clones all showed the nucleotide sequence of NR8. Therefore, the sequence of NR8 was not included in the six clones whose nucleotide sequences were analyzed here. The 3'-UTO of the NR8 cDNA clone whose sequence was determined was the same as the 3'-UTR terminal sequence (3UTR-1) of NR8 obtained by 3'-RACE. The presence of a cDNA clone in which a poly A tail is added (3UTR-2) to the site extended by 483 bp and a cDNA clone in which a poly A tail is added (3UTR-3) to the site extended by 2397 bp Was confirmed. On the other hand, of the two clones whose sequences were determined as described above, both contained the base sequence of 3UTR-3. Table 10 below summarizes the sequences of the 3'-terminal untranslated region retained by the cDNA clones obtained thus far. In addition, the nucleotide sequences of 3UTR-1, 3UTR-2 and 3UTR-3 as calculated from the translation termination codon of the NR8 cDNA sequence are represented by SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively. It was shown to.
さらに NR8 ? cDNA配列の翻訳終止コ ドンから起算した場合の 3UTR-B1、及び 3 UTR-B3の塩基配列を、 それそれ配列番号： 2 6および配列番号： 2 7に示した c 表 1 0 Further NR8 a nucleotide sequence of 3UTR-B1, and 3 UTR-B3 in the case of counting from the translation termination codon of the cDNA sequence, it it SEQ ID NO: 2 6 and SEQ ID NO: 2 7 c Table 1 0 shown in
NR8 cDNAクローン 3'-UTR配列 NR8 cDNA clone 3'-UTR sequence
NR8ひ 3UTR-1 NR8 3UTR-1
NR8 ? 3UTR-B1 , 3UTR-B3  NR8? 3UTR-B1, 3UTR-B3
NR8 r 3UTR-1, 3UTR-2, 3UTR-3  NR8 r 3UTR-1, 3UTR-2, 3UTR-3
以上によって得られた塩基配列より、 NR8 の遺伝子転写産物は選択的スプラ イシングの相違のみならず、 3'末端非翻訳領域配列の長さによっても種々の異 なるサイズをコードし得ることが判明した。 このことは、 ノーザン解析によつ て検出された、 種々のサイズを示す転写産物の存在を十分に説明しうる。 Based on the nucleotide sequences obtained as described above, it was found that the NR8 gene transcript could encode various different sizes depending on the length of the 3′-terminal untranslated region sequence as well as the difference in alternative splicing. . This may fully explain the presence of transcripts of various sizes, detected by Northern analysis.
[実施例 6 ] リガンドスクリーニング 6 ) - 1 NR8キメラ受容体の構築 [Example 6] Ligand screening 6) -1 Construction of NR8 chimeric receptor
NR8に特異的に結合し得るリガンド、即ち新規へモポェチンを検索するための スクリ一ニング系を構築した。先ず最初に NR8ひの細胞外領域（配列番号： 1の アミノ酸配列； 1位の Metから、 2 2 8位の Gluまで） をコードする cDNA配列を P CRによって増幅し、この DNA断片を既知のへモポェチン受容体の細胞膜貫通領域、 及び細胞内領域をコードする DNA断片とィンフレームで結合させることによつ て、 キメラ受容体をコードする融合配列を作製した。 ここで、 パートナーとな る既知へモポェチン受容体として、 前述のようにいくつかの候補が挙げられた が、 その中からヒ ト TP0受容体（Human MPL- P )を選択して用いた。 すなわち、 ヒ ト TP0受容体の細胞膜貫通領域を含む細胞内領域をコードする DNA配列を PCRに よって増幅した後、 NR8ひの細胞外領域をコ一ドする cDNA配列と、 インフレーム で結合させ、 哺乳動物細胞で発現可能なプラスミ ドベクターに挿入した。 構築 した発現べクタ一を pEF- NR8/TP0- Rと名付けた。 また、 構築した NR8/TP0-Rキメ ラ受容体の構造の模式図を図 1 4に、 さらに、 構築したキメラ受容体の塩基配 列とそれがコードする発現可能なアミノ酸配列をそれそれ配列番号： 1 3およ び 1 4に示す。 NR8/TP0- Rキメラ受容体発現べクタ一はブラス トサイジン S耐性 遺伝子を含む発現べクタ一 pSV2bsr (科研製薬株式会社製） と共に増殖因子依存 性細胞株 Ba/F3に導入して強制発現させた後、 8〃g/mlの塩酸ブラストサイジン S (科研製薬株式会社製） と IL3の共存下で培養することにより遺伝子導入細胞 を選別した。 得られたキメラ受容体導入細胞を IL- 3非存在下に切り替え、 標的 リガンドを含むことが期待される材料を添加して培養することにより、 NR8と特 異的に結合するリガンドが存在する場合にのみ生存/増殖可能であることを利 用したスクリーニングが実施可能である。  A screening system was constructed to search for a ligand capable of specifically binding to NR8, ie, a novel hemopoietin. First, the cDNA sequence encoding the extracellular region of NR8 (amino acid sequence of SEQ ID NO: 1; from Met at position 1 to Glu at position 228) was amplified by PCR, and this DNA fragment was A fusion sequence encoding a chimeric receptor was prepared by binding in frame with a DNA fragment encoding the transmembrane region and the intracellular region of the hemopoietin receptor. Here, as mentioned above, several candidates were listed as known partners of the known hemopoietin receptor. From these, the human TP0 receptor (Human MPL-P) was selected and used. That is, after amplifying by PCR the DNA sequence encoding the intracellular region including the transmembrane region of the human TP0 receptor, it is combined in-frame with the cDNA sequence encoding the extracellular region of NR8, It was inserted into a plasmid vector that can be expressed in mammalian cells. The constructed expression vector was named pEF-NR8 / TP0-R. Fig. 14 shows a schematic diagram of the structure of the constructed NR8 / TP0-R chimera receptor. Furthermore, the base sequence of the constructed chimeric receptor and the expressible amino acid sequence encoded by it are shown in SEQ ID NOs. : Shown in 13 and 14. The NR8 / TP0-R chimeric receptor expression vector was introduced into the growth factor-dependent cell line Ba / F3 together with the expression vector pSV2bsr (manufactured by Kaken Pharmaceutical Co., Ltd.) containing the blasticidin S resistance gene and forcedly expressed. Thereafter, the transduced cells were selected by culturing in the presence of 8 µg / ml blasticidin hydrochloride S (manufactured by Kaken Pharmaceutical Co., Ltd.) and IL3. When the resulting chimeric receptor-introduced cells are switched to the absence of IL-3, and a material that is expected to contain the target ligand is added and cultured, and a ligand that specifically binds to NR8 is present. Screening using viability / proliferation is only feasible.
6 )-2 NR8/IgG卜 Fc可溶性融合蛋白質の調製  6) -2 Preparation of NR8 / IgG Fc soluble fusion protein
細胞膜結合型リガンドの探索、 あるいは B IAcore (Pharmac ia社） やウェスト ウエスタン法による可溶性リガンドの検出に利用すべく NR8/ IgGl- Fc可溶性融 合蛋白質の調製を行った。 5 )-1項で調製した NR8ひの細胞外領域 （アミノ酸配 列； 1位の Metから、 2 2 8位の Gluまで） をコ一ドする DNA断片をヒト免疫グロ プリン IgGlの Fc領域をコードする DNA断片とィンフレームで結合させることに よって、 該可溶性融合蛋白質をコードする融合配列を作製した。 構築した NR8/ IgGl- Fcがコードする可溶性融合蛋白質の構造の模式図を図 1 4に、 さらに、そ の塩基配列とそれがコードする発現可能なアミノ酸配列をそれそれ配列番号： 1 5および 1 6に示す。 該融合遺伝子断片を哺乳動物細胞で発現可能なプラス ミ ドベクターに挿入し、構築した発現べクタ—を pEF-NR8/ IgGl- Fcと名付けた。 この pEF- NR8/IgGl- Fcを哺乳動物細胞に強制発現させ、安定した遺伝子導入細胞 を選択した後、 その培養上清に分泌される当該リコンビナント蛋白質を、 抗ヒ ト IgGl- Fc抗体を用いた免疫沈降、あるいはァフィ二ティーカラム等により精製 することが可能である。 Use NR8 / IgG1-Fc soluble fusion to search for cell membrane-bound ligands or to detect soluble ligands by BIAcore (Pharmacia) or West Western method. A synthetic protein was prepared. 5) The DNA fragment encoding the extracellular region (amino acid sequence; from Met at position 1 to Glu at position 228) of NR8 prepared in section -1 was ligated to the Fc region of human immunoglobulin IgGl. A fusion sequence encoding the soluble fusion protein was prepared by in-frame binding to the encoding DNA fragment. A schematic diagram of the structure of the soluble fusion protein encoded by the constructed NR8 / IgGl-Fc is shown in Fig. 14, and its nucleotide sequence and the expressible amino acid sequence encoded by it are shown in SEQ ID NOS: 15 and 1, respectively. See Figure 6. The fusion gene fragment was inserted into a plasmid vector that can be expressed in mammalian cells, and the constructed expression vector was named pEF-NR8 / IgGl-Fc. After forcibly expressing this pEF-NR8 / IgGl-Fc in mammalian cells and selecting stable transfected cells, the recombinant protein secreted in the culture supernatant was purified using an anti-human IgGl-Fc antibody. It can be purified by immunoprecipitation or affinity column.
6 )-3 NR8 ?の発現系構築とリコンピナント 蛋白質の精製  6) -3 Construction of NR8? Expression system and purification of recombinant protein
細胞膜結合型リガンドの探索、 あるいは BIAcore (Pharmacia社） やウェスト ウエスタン法による可溶性リガンドの検出に利用すべく リコンビナント NR8 ? 蛋白質の調製を行った。 NR8 3 cDNAのアミノ酸コーディング配列を用い、 終止 コドンを点変異によって任意のアミノ酸残基をコ一ドする塩基配列に置換した 後、 インフレ一ムで FLAGペプチドをコードする塩基配列に結合させた。 その結 合断片を哺乳動物細胞で発現可能なブラスミ ドベクタ一に挿入し、 構築した発 現べクタ一を PEF-B0S/NR8 ? FLAGと名付けた。 構築した発現べクタ一中の挿入 断片 NR8 ? FLAGの構造の模式図を図 1 4に示す。 さらに FLAGの塩基配列 と、 それがコードする発現可能なアミノ酸配列を配列番号： 1 7および 1 8に 示す。 この PEF-B0S/NR8 ? FLAGを哺乳動物細胞に強制発現させ、 安定した遺伝 子導入細胞を選択した後、 その培養上清に分泌される当該リコンビナント蛋白 質を、 抗 FLAGペプチド抗体を用いて免疫沈降を行うことが可能であり、 あるい はァフィ二ティ一カラム等により精製することが可能である。 [実施例 7 ] マウス NR8 (mNR8) 遺伝子の単離 Recombinant NR8 protein was prepared to search for cell membrane-bound ligand or to detect soluble ligand by BIAcore (Pharmacia) or West Western method. Using the amino acid coding sequence of the NR83 cDNA, the termination codon was replaced with a nucleotide sequence encoding any amino acid residue by point mutation, and then linked to the nucleotide sequence encoding the FLAG peptide in in-frame. The ligated fragment was inserted into a plasmid vector that can be expressed in mammalian cells, and the constructed expression vector was named PEF-B0S / NR8-FLAG. A schematic diagram of the structure of the inserted fragment NR8-FLAG in the constructed expression vector is shown in FIG. Furthermore, the nucleotide sequence of FLAG and the expressible amino acid sequence encoded thereby are shown in SEQ ID NOs: 17 and 18. After forcibly expressing the PEF-B0S / NR8-FLAG in mammalian cells and selecting stable transfected cells, the recombinant protein secreted into the culture supernatant is immunized with an anti-FLAG peptide antibody. Sedimentation can be performed, or purification can be performed using an affinity column or the like. [Example 7] Isolation of mouse NR8 (mNR8) gene
7-1 ) ヒト NR8プライマ一を用いたマウス相同遺伝子の単離  7-1) Isolation of mouse homologous gene using human NR8 primer
ヒト NR8の全長 cDNAを単離する際に用いたォリゴヌクレオチドプライマ一で ある、 センス側 （下流方向） の NR8-SN1 と NR8-SN2 (配列番号： 9および 1 0 )、 またアンチセンス側 （上流方向） の NR8- AS1 と NR8- AS2 (配列番号： 1 1およ び 1 2 ) を利用して、 異種間交叉 PCRクローニングを試みた。 上記のヒト NR8 プライマーの組合わせにより、 4通りのプライマ一セッ 卜が構成可能である。 すなわち、 [NR8-SN1 対腦 -AS1 ]ヽ [NR8-SN1 対 NR8-AS2]、 [NR8-SN 対蘭 - AS1 ]、 及び [NR8-SN2 対 NR8- AS2] の組合わせを用い、 マウス脳 cDNA(Clontech #7450-1 ), 及びマウス精巣 cMA(C lontech#7455-l )ライブラリ一を銪型として 実施することによって、 交叉 PCR産物の増幅を期待した。 PCR反応には Advant age cDNA Polymerase Mix ( Clontech#8417-l )を使用した。 Perkin Elmer G ene Amp PCR System 2400 サ一マルサイクラ一を使用し、 下記のサイクル条件 にて実施することで、 同受容体のマウス相同遺伝子をコードし得る部分塩基配 列の増幅を試みた。  The oligo nucleotide primers used to isolate the full-length human NR8 cDNA, NR8-SN1 and NR8-SN2 on the sense side (downstream direction) (SEQ ID NOS: 9 and 10), and the antisense side ( Heterologous crossover PCR cloning was attempted using NR8-AS1 and NR8-AS2 (upstream direction) (SEQ ID NOS: 11 and 12). By combining the above human NR8 primers, four types of primer sets can be constructed. That is, using the combination of [NR8-SN1 vs. brain -AS1] ヽ [NR8-SN1 vs. NR8-AS2], [NR8-SN vs. Netherlands -AS1], and [NR8-SN2 vs. NR8-AS2], the mouse brain Amplification of the cross-PCR product was expected by performing the cDNA (Clontech # 7450-1) and mouse testis cMA (Clontech # 7455-l) library type 1 as type I. Advantage cDNA Polymerase Mix (Clontech # 8417-l) was used for the PCR reaction. By using a Perkin Elmer Gene Amp PCR System 2400 Thermal Cycler under the following cycle conditions, an attempt was made to amplify a partial base sequence capable of encoding a mouse homologous gene of the receptor.
即ち、 交叉 PCR条件は、 94°Cで 4分、 「94°Cで 20秒、 72°Cで 1分」 を 5サイ クル、 「94°Cで 20秒、 70°Cで 1分」 を 5サイクル、 「94°Cで 20秒、 68°Cで 1分」 を 28サイクル、 72。Cで 4分、 および 4°Cにて終結、 である。  That is, the cross-PCR conditions were as follows: 4 minutes at 94 ° C, 5 cycles of `` 20 seconds at 94 ° C, 1 minute at 72 ° C '', 5 cycles of `` 20 seconds at 94 ° C, 1 minute at 70 ° C '' 5 cycles, 28 cycles of “20 seconds at 94 ° C, 1 minute at 68 ° C”, 72 cycles. Terminate at 4 ° C for 4 minutes and at 4 ° C.
この結果、 図 1 5に示す通り、 何れのプライマ一セッ トを用いた場合でも交 叉 PCR産物の増幅が認められた。また、 マウス精巣 cDNAを鍊型として用いた場 合より、マウス脳 cDNAを錡型としたときの方がより鮮明な増幅産物を得ること ができた。  As a result, as shown in FIG. 15, amplification of the cross-PCR product was observed using any of the primer sets. In addition, a clearer amplification product could be obtained when the mouse brain cDNA was used as type III than when the mouse testis cDNA was used as type III.
7-2 ) NR8に対応するマウス相同遺伝子の部分塩基配列の決定  7-2) Determination of partial nucleotide sequence of mouse homologous gene corresponding to NR8
7-1 )で得られた増幅産物のうち、マウス脳 cDNA由来の産物を pGEM- T Easy v ector (Promega #A1360 ) にサブクロ一ニングし、 塩基配列を決定した。 PCR 産物の pGEM- T Easy vector への組換えは、 T4 DNA Ligase (Promega#A1360 ) によって、 4 °C /12時間の反応をおこなった。 PCR 産物と pGEM-T Easy vecto rの遺伝子組換え体は、 大腸菌株 DH5ひ（Toyobo #DNA_903 )を形質転換すること によって得られた。 また、 遺伝子組換え体の選別には、 Insert Check Ready B lue ( Toyobo#PIK- 201 )を用いた。 さらに、 塩基配列の決定には、 BigDye Termi nator Cyc le Sequenc ing Ready Reaction Kit (ABI/Perkin Elmer#4303154)を 使用し、 AB I PRISM 377 DNA Sequencer によって解析をおこなった。 独立する 8クローンの遺伝子組換え体に対し、 全ィンサート断片の塩基配列を決定した 結果、 同一の転写産物に由来する塩基配列が得られ、 それらが共に mNR8の部分 塩基配列である事を認めた。 得られた部分塩基配列を配列番号 ： 2 8に示す。 Among the amplification products obtained in 7-1), a mouse brain cDNA-derived product was subcloned into pGEM-T Easy vector (Promega # A1360) to determine the nucleotide sequence. Recombination of the PCR product into pGEM-T Easy vector is performed using T4 DNA Ligase (Promega # A1360). The reaction was carried out at 4 ° C for 12 hours. The PCR product and the recombinant of pGEM-T Easy vector were obtained by transforming E. coli strain DH5 (Toyobo # DNA_903). In addition, the recombinants were selected using Insert Check Ready Blue (Toyobo # PIK-201). Furthermore, the nucleotide sequence was determined using the ABI PRISM 377 DNA Sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI / Perkin Elmer # 4303154). As a result of determining the nucleotide sequence of all the insert fragments in eight independent clones of the recombinant, a nucleotide sequence derived from the same transcript was obtained, and it was confirmed that both were partial nucleotide sequences of mNR8 . The obtained partial base sequence is shown in SEQ ID NO: 28.
7-3 ) マウス NR8遺伝子特異的オリゴヌクレオチドプライマ一の設計  7-3) Design of oligonucleotide primers specific for mouse NR8 gene
7-2 )で得られた mNR8の部分塩基配列をもとに、マウス NR8に特異的なオリゴ ヌクレオチドプライマ一の設計をおこなった。 下に配列を記す通り、 センス側 (下流方向） に mNR8-SN3を、 またアンチセンス側 （上流方向） に mNR8- AS3を それそれ合成した。 プライマ一の合成には、 AB I社の 394 DNA/RNA Synthesize r を使用し、 5'-末端トリチル基付加条件にて実施した。その後、 OPC column (A B I#400771 ) にて、完全長の合成産物を精製した。これらプライマーは後述の 5' - RASE法、 及び 3'- RACE法に提供した。  Based on the partial nucleotide sequence of mNR8 obtained in 7-2), an oligonucleotide primer specific to mouse NR8 was designed. As shown below, mNR8-SN3 was synthesized on the sense side (downstream) and mNR8-AS3 was synthesized on the antisense side (upstream). The primer was synthesized using ABI 394 DNA / RNA Synthesizer under the condition of adding a 5'-terminal trityl group. Thereafter, the full-length synthetic product was purified using an OPC column (ABI # 400771). These primers were provided for the 5'-RASE and 3'-RACE methods described below.
mNR8-SN3 ； 5 ' - TCC AGG CGC TCA GAT TAC GAA GAC CCT GCC -3 ' (配列番号：mNR8-SN3; 5'-TCC AGG CGC TCA GAT TAC GAA GAC CCT GCC -3 '(SEQ ID NO:
2 9 ) 2 9)
mNR8-AS3 ； 5 ' - ACT CCA GGT CCC CTG GTA GGA GGA GCC AGG -3 ' (配列番号：mNR8-AS3; 5'-ACT CCA GGT CCC CTG GTA GGA GGA GCC AGG-3 '(SEQ ID NO:
3 0 ) 3 0)
7-4) 5'- RACE法による N末端 cDNAのクロ一ニング  7-4) Cloning of N-terminal cDNA by 5'-RACE method
mNR8の全長 cDNA を単離するために、 NR8-AS2 プライマ一（配列番号： 1 2 ) を一次 PCRに用い、 また、 前項の mNR8- AS3プライマー （配列番号： 3 0 ) を 二次 PCRに用いて 5，-RACE PCRを試みた。 錶型として Mouse Brain arathon-R eady cDNA Library ( Clontech#7450- 1 )を用い、 PCR 実験には Advantage cDNA bU In order to isolate the full-length mNR8 cDNA, the NR8-AS2 primer (SEQ ID NO: 12) was used for primary PCR, and the mNR8-AS3 primer (SEQ ID NO: 30) described in the previous section was used for secondary PCR. 5, -RACE PCR was attempted. Mouse Brain arathon-Ready cDNA Library (Clontech # 7450-1) was used as type 錶, and Advantage cDNA was used for PCR experiments. bU
Polymerase Mix を使用した。 Perkin Elmer Gene Amp PCR System 2400 サー マルサイクラ一を使用し、 下記の PCR 条件で実施した結果、 2種類のサイズの 異なる PCR産物が得られた。 Polymerase Mix was used. Using the Perkin Elmer Gene Amp PCR System 2400 Thermal Cycler under the following PCR conditions, PCR products of two different sizes were obtained.
一次 PCRの条件は、 94°Cで 4分、 「94°Cで 20秒、 72°Cで 100秒」 を 5サイク ル、 「94°Cで 20秒、 70°Cで 100秒」 を 5サイクル、 「94°Cで 20秒、 68°Cで 100 秒」 を 28サイクル、 72°Cで 3分、 および 4°Cにて終結である。  Primary PCR conditions were 4 minutes at 94 ° C, 5 cycles of `` 20 seconds at 94 ° C, 100 seconds at 72 ° C '', and 5 cycles of `` 20 seconds at 94 ° C, 100 seconds at 70 ° C ''. The cycle, "20 seconds at 94 ° C, 100 seconds at 68 ° C", is completed at 28 cycles, 3 minutes at 72 ° C, and 4 ° C.
二次 PCRの条件は、 94°Cで 4分、 「94°Cで 20秒、 70°Cで 100秒」 を 5サイク ル、 「94°Cで 20秒、 68°Cで 100秒」 を 25サイクル、 72°Cで 3分、 および 4°Cに て終結である。  The conditions for secondary PCR were 4 minutes at 94 ° C, 5 cycles of `` 20 seconds at 94 ° C, 100 seconds at 70 ° C '', and 5 cycles of `` 20 seconds at 94 ° C and 100 seconds at 68 ° C ''. 25 cycles, 3 minutes at 72 ° C, and termination at 4 ° C.
得られた 2種類の PCR 産物は共に、前記同様、 pGEM- T Easy vectorにサブク ローニングし、塩基配列を決定した。 PCH 産物の pGEM-T Easy vectorへの組換 えは、 T4 DNA Ligase によって、 4°C /12 時間の反応をおこなった。 PCR 産物 と pGEM-T Easy vectorの遺伝子組換え体は、大腸菌株 DH5ひを形質転換するこ とによって得られた。 また、 遺伝子組換え体の選別も前述と同様に、 Insert C heck Ready Blueを用いた。 塩基配列の決定においても、 BigDye Terminator C ycle Sequenc ing Ready Reaction Kitを使用し、 AB I PRI SM 377 DNA Sequence r によって解析を実施した。 独立する 8クローンの遺伝子組換え体に対し、 全 インサート断片の塩基配列を決定した結果、 塩基対の長さ、 及び配列の相違に より、 それそれ 4クローンずつの 2種類のグループに区別することができた。 これは、 選択的スプライシングに起因する産物の相違であり、 この得られた配 列が、 双方共に完全長 mNil8 cDNAクローンの N末端配列を含んでいることを認 めた。 ここで、 プロリンに富む領域 （Pro- rich領域） をコードするェキソンを 含む長い 0RFを保有する cDNAクローンを mNR8ァと命名し、 Pro-rich領域を保 有しない短い 0RFをコ一ドする cDNAクローンを mNR8 βと命名した。 これらの cDNAクローンはそれそれ、 ヒト NR8ァ、 及びヒト NR8 ?の異生物種相同遺伝子 に相当する。 7-5 ) 3 -RACE法による C末端 cDNAのクローニング Both of the obtained two PCR products were subcloned into the pGEM-T Easy vector as described above, and the nucleotide sequences were determined. The recombination of the PCH product into the pGEM-T Easy vector was performed at 4 ° C for 12 hours using T4 DNA Ligase. PCR products and recombinants of the pGEM-T Easy vector were obtained by transforming E. coli strain DH5. In addition, for the selection of transgenic plants, Insert Check Ready Blue was used in the same manner as described above. In the determination of the nucleotide sequence, the analysis was carried out by ABI PRI SM 377 DNA Sequencer using the BigDye Terminator Cycle Sequencing Ready Reaction Kit. The nucleotide sequence of all insert fragments was determined for 8 independent clones of the recombinant, and based on differences in base pair length and sequence, each clone was divided into two groups of 4 clones each. Was completed. This was a product difference due to alternative splicing, confirming that both resulting sequences contained the N-terminal sequence of the full length mNil8 cDNA clone. Here, a cDNA clone having a long 0RF containing an exon encoding a proline-rich region (Pro-rich region) is designated as mNR8a, and a cDNA clone encoding a short 0RF without a pro-rich region. Was named mNR8β. These cDNA clones correspond to the human NR8a and the heterologous gene of human NR8 ?, respectively. 7-5) Cloning of C-terminal cDNA by 3-RACE method
mNR8の全長 cDNA を単離するために、 NR8-SN1プライマ一 （配列番号： 9 ) を一次 PCRに用い、 また、 mNR8-SN3プライマ一 （配列番号： 2 9 ) を二次 PCR に用いて 3 -RACE PCRを試みた。 鍊型として Mouse Brain Marathon-Ready cDN A Libraryを用い、 PCR 実験には Advantage cDNA Polymerase Mix を使用した。 Perkin Elmer Gene Amp PCR System 2400サーマルサイクラ一を使用し、前記 同様の PCR 条件で実施した結果、 単一のサイズを示す PCR産物が得られた。得 られた PCR 産物は、 前記 7- 2 )の記載に従い、 pGEM- T Easy vectorにサブクロ 一二ングし、 塩基配列を決定した。 独立する 4クローンの遺伝子組換え体に対 し、 全インサート断片の塩基配列の決定をおこなった結果、 完全長 mNR8 cDNA クローンの C末端配列を含んでいることを認めた。 この 3'RACE- PCRの結果、 決 定できた塩基配列と、前記 7- 4)において決定した 5'RACE-PCR産物の塩基配列と を総合することによって、 最終的に完全長 mNR8ァ、 及び完全長 mNR8 ? cDNAの 全塩基配列を決定した。 決定した mNR8ァ cDNAの塩基配列及びそれがコードす るアミノ酸配列をそれそれ配列番号： 2 2および 2 1に示す。 また、 決定した mNR8^ cDNA の塩基配列及びそれがコ一ドするアミノ酸配列を配列番号： 2 0 および 1 9に示す。  To isolate the full-length mNR8 cDNA, the NR8-SN1 primer (SEQ ID NO: 9) was used for primary PCR, and the mNR8-SN3 primer (SEQ ID NO: 29) was used for secondary PCR. -RACE PCR was attempted. Mouse Brain Marathon-Ready cDNA Library was used as type 鍊, and Advantage cDNA Polymerase Mix was used for PCR experiments. Using a Perkin Elmer Gene Amp PCR System 2400 thermal cycler under the same PCR conditions as above, a PCR product showing a single size was obtained. The obtained PCR product was subcloned into the pGEM-T Easy vector according to the description in 7-2), and the nucleotide sequence was determined. The nucleotide sequence of all the insert fragments was determined for four independent recombinants, and it was confirmed that they contained the C-terminal sequence of the full-length mNR8 cDNA clone. By combining the nucleotide sequence determined as a result of the 3′RACE-PCR with the nucleotide sequence of the 5′RACE-PCR product determined in the above 7-4), the final full-length mNR8α, and The entire nucleotide sequence of the full-length mNR8 cDNA was determined. The determined nucleotide sequence of the mNR8 cDNA and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 22 and 21, respectively. Also, the determined nucleotide sequence of mNR8 ^ cDNA and the amino acid sequence encoded thereby are shown in SEQ ID NOs: 20 and 19.
ァミノ酸配列をヒトとマウスの NR8間において比較したところ、 NR8ァでは 9 8. 9% の高い相同性が認められ、一方 NR8 5においても 97.2%の相同性を認めた。 この結果は、 同受容体遺伝子が生物種間を越えて重要な機能責任を担っている 可能性を強く示唆するものである。 ヒト及びマウス NR8 ?のアミノ酸配列を比 較した結果を図 1 6に記載した。 また、 ヒ ト及びマウス NR8ァのアミノ酸配列 を比較した結果を図 1 7に記載した。  When the amino acid sequences were compared between human and mouse NR8, a high homology of 98.9% was observed in NR8a, while a homology of 97.2% was also observed in NR85. This result strongly suggests that the receptor gene may have important functional responsibilities across species. FIG. 16 shows the results of comparing the amino acid sequences of human and mouse NR8 ?. The results of comparing the amino acid sequences of human and mouse NR8a are shown in FIG.
最終的に単離した mNR8ァ、 及び mNR8 ?の完全長 cDNAの両者はヒト NR8同様 の選択的スプライシングによって、 538 アミノ酸からなる細胞膜貫通型受容体 蛋白と、 144 アミノ酸からなる可溶性受容体様蛋白をそれそれコード可能であ つた。 mNR8ァの特徴として以下の構造が認められる。 先ず最初にアミノ酸番号 1位の Metから 19位の Glyまでが典型的な分泌シグナル配列であると予測され る。 ここで、 1位の Metよりマイナス 13位の位置に、 ィンフレームの終止コド ンが存在するため、 この Met残基が翻訳開始コドンであると推定される。 次に 25位の Cysから 35位の Cys残基までが、 典型的なリガンド結合部位配列であ り、さらに 65位と 109位の Cys残基は他のへモポェチン受容体メンバ一にもよ く保存された Cys残基の繰り返し構造を示す。続いて 120位、 及び 122位と 12 3位に連続する Pro残基によって、 Pro-rich領域が保存されており、 さらに、 2 14位の Trpから 218位の Ser残基までに典型的な WSXWS-box (WSモチーフ）が 認められる。これら細胞外領域における構造的特徴に続き、 233位の Glyから 2 55位の Leu残基までの 23ァミノ酸に典型的な細胞膜貫通ドメインを保有して いる。 さらに、 その直後の細胞内領域における 271位、 及び 273位の Pro残基 は、 他のへモポェチン受容体メンバーにもよく保存された Box-1コンセンサス 配列 （PXP モチーフ） であり、 ここがシグナル伝達に深く関与すると考えられ る。以上のように mNR8ァはへモポェチン受容体メンバ一の特徴を充分に満足さ せる。 Both the finally isolated mNR8a and mNR8? Full-length cDNAs, through alternative splicing similar to human NR8, produce a 538 amino acid transmembrane receptor protein and a 144 amino acid soluble receptor-like protein. Each one is codeable I got it. The following structures are recognized as features of mNR8a. First, a typical secretory signal sequence from Met at amino acid number 1 to Gly at position 19 is predicted. Here, an inframe stop codon is present at a position minus 13 relative to the Met at position 1, so that this Met residue is presumed to be a translation initiation codon. Next, Cys at position 25 to Cys residue at position 35 is a typical ligand binding site sequence, and the Cys residues at positions 65 and 109 are common to other hemopoietin receptor members. 1 shows the conserved Cys residue repeat structure. The Pro-rich region is subsequently conserved by Pro residues at positions 120 and 122 and 123, and a typical WSXWS from Trp at position 2 14 to Ser at position 218. -box (WS motif) is recognized. Following these structural features in the extracellular region, it possesses a transmembrane domain typical of 23 amino acids from Gly at position 233 to Leu residue at position 255. In addition, the Pro residues at positions 271 and 273 in the intracellular region immediately after that are Box-1 consensus sequences (PXP motifs) that are well conserved among other hemopoietin receptor members, and this is signal transduction. It is thought to be deeply involved in As described above, mNR8a fully satisfies the characteristics of one member of the hemopoietin receptor.
一方、 mM8 ?においては、 上記細胞外領域における構造的特徴のうち、 Pro - rich 領域をコ一ドするェキソン配列が選択的スプライシングによってスキヅ プされ、 WSモチーフをコードする次のェキソン部位に直結する。 しかし、 フレ 一ムシフ トによって、 WSXWS- box配列は読み枠から外れ、 144位の Leu残基まで をコードした後、 次の終止コドンによって翻訳フレームが終結する。 これによ つて、 細胞膜貫通ドメインを保持しない、 可溶性へモポェチン受容体様蛋白を コードしている。  On the other hand, in mM8 ?, among the structural features in the extracellular region, the exon sequence coding for the pro-rich region is skipped by alternative splicing and directly connected to the next exon site encoding the WS motif. . However, the frame shift shifts the WSXWS-box sequence out of reading frame, encoding up to Leu residue at position 144, and then terminating the translation frame with the next stop codon. This encodes a soluble hemopoietin receptor-like protein that does not have a transmembrane domain.
[実施例 8 ] マウス NR8遺伝子の発現解析  [Example 8] Expression analysis of mouse NR8 gene
8-1 ) RT-PCR法によるマウス NR8遺伝子発現様態の解析  8-1) RT-PCR analysis of mouse NR8 gene expression
各マウス臓器における NR8遺伝子の発現分布、 及び、 遺伝子発現様態を解析 bo Analysis of NR8 gene expression distribution and gene expression in each mouse organ bo
するために、 RT- PCR解析法による mMAの検出を行った。 RT-PCR解析に用いる プライマーとして、センス側（下流方向）プライマ一に NR8-SN1プライマー（配 列番号： 9 ) を選択し、 アンチセンス側 （上流方向） プライマーに NR8- AS1 Pr imerを選択した。 錶型として、 Mouse Multiple Tissuue cDNA Panel ( Clontec h #K1423-1 )を用いた。 PCRには Advantage cDNA Polymerase Mix ( Clontech#8 417-1 ) を用い、 Perkin Elmer Gene Amp PCR System 2400サーマルサイクラ一 を使用した。 PCR 反応は下記のサイクル条件にて実施することで、 標的遺伝子 の増幅を試みた。 In order to perform this, mMA was detected by RT-PCR analysis. As primers used for RT-PCR analysis, NR8-SN1 primer (SEQ ID NO: 9) was selected as the primer on the sense side (downstream direction), and NR8-AS1 Primer was selected as the primer on the antisense side (upstream direction). . The mouse Multiple Tissuue cDNA Panel (Clontec h # K1423-1) was used as type 錶. PCR was performed using an Advantage cDNA Polymerase Mix (Clontech # 8 417-1) and a Perkin Elmer Gene Amp PCR System 2400 thermal cycler. The PCR reaction was performed under the following cycle conditions to try to amplify the target gene.
PCR条件は、 94°Cで 4分、 「94°Cで 20秒、 72°Cで 1分」 を 5サイクル、 「94°C で 20秒、 70°Cで 1分」 を 5サイクル、 「94°Cで 20秒、 68°Cで 1分」 を 24サイ クル、 72°Cで 3分、 および 4°Cにて終結、 である。  The PCR conditions were 4 cycles at 94 ° C, 5 cycles of `` 20 seconds at 94 ° C, 1 minute at 72 ° C '', 5 cycles of `` 20 seconds at 94 ° C, 1 minute at 70 ° C '', 20 cycles at 94 ° C, 1 minute at 68 ° C ”are 24 cycles, 3 minutes at 72 ° C, and end at 4 ° C.
RT - PCRの結果を図 1 8に示す通り、 精巣、 及び 17 日目胚において同遺伝子の 強発現が検出されたほか、 解析をおこなった全てのマウス臓器、 及び組織由来 の mRNAにおいて構成的な遺伝子発現が認められた。また解析に使用した全ての 踌型に対して、 マウス G3PDHプライマーを用い上記 PCR条件にてハウスキービ ング遺伝子 G3PDHの発現を検出することで、予め錶型 mRNAのコピー数がサンプ ル間で Normal ize (標準化） されていることを確認している。 また、 ここで、 検出された RT- PCR増幅産物のサイズは、 320 bpであり、 これは決定した塩基 配列から計算されるサイズと一致する。 従ってこれらは、 マウス NR8特異的な PCR増幅反応による産物であると考えられた。このことを更に確認するために、 17日目胚において増幅された PCR産物を前記 7-2 )に従い、 pGEM- T Easy vecto rにサブクローニングし、 塩基配列を解析した。 その結果マウス NR8の部分塩 基配列であることを認め、 非特異的な PCR増幅による産物である可能性を否定 した。  As shown in Fig. 18, RT-PCR results showed that strong expression of the gene was detected in testis and day 17 embryos, and that constitutive mRNA was analyzed in all mouse organs and tissues analyzed. Gene expression was observed. In addition, by detecting the expression of the housekeeping gene G3PDH under the above PCR conditions using the mouse G3PDH primer for all the 踌 type used in the analysis, the copy number of type mRNA mRNA was previously normalized between samples. Standardized). Also, the size of the RT-PCR amplification product detected here was 320 bp, which matches the size calculated from the determined base sequence. Therefore, these were considered to be products of mouse NR8-specific PCR amplification reaction. To further confirm this, the PCR product amplified in the 17-day embryo was subcloned into pGEM-T Easy vector according to the above 7-2), and the nucleotide sequence was analyzed. As a result, it was confirmed to be a partial nucleotide sequence of mouse NR8, and the possibility of being a product of non-specific PCR amplification was denied.
8-2 ) ノーザンプロッティング法によるマウス NR8遺伝子の発現様態解析 各マウス臓器における NR8の遺伝子発現様態の解析と、 NR8転写サイズの同 定を目的として、 ノーザンプロッティング法による遺伝子発現解析を試みた。 ブロッ 卜には Mouse Multiple Tissue Northern Blot ( Clontech #7762-1 )を 使用した。 プローブには前記 7-4)にて得られた、 5'-RACE産物のうち mNR8 ? c DM断片を用いた。 プローブの調製は、 Mega Prime Kit (Amersham, cat#RPN16 07)を使用し [ひ-32 P]dCTP (Amersham, cat#AA0005 )によってラジオアイソトー プ標識した。ハイブリダイゼ一シヨンには Express Hyb-ridization Solution (Clontech#8015-2 )を用い、 68°C/30分のプレハイプリダイゼーシヨンの後、 熱 変性させた標識プローブを加え、 68°C/ 16時間のハイブリダィゼ一シヨンを実 施した。 下記の条件にて洗浄をおこなった後、 Imaging Plate (FUJI#BAS-I I I ) に露光させ、 Image Analyzer (FUJIX, BAS-2000 I I )によって、 マウス NR8特異 的なシグナルを検出した。 8-2) Analysis of expression pattern of mouse NR8 gene by Northern plotting Analysis of gene expression pattern of NR8 in each mouse organ and analysis of NR8 transcript size For the purpose of determination, gene expression analysis by Northern plotting was attempted. Mouse Multiple Tissue Northern Blot (Clontech # 7762-1) was used for the blot. The mNR8-cDM fragment of the 5'-RACE product obtained in 7-4) was used as a probe. Preparation of probe, Mega Prime Kit (Amersham, cat # RPN16 07) using the shed - 32 P] dCTP (Amersham, cat # AA0005) were radio-eye Soto flop labeled with. Express Hyb-ridization Solution (Clontech # 8015-2) is used for hybridization, and after pre-hybridization at 68 ° C / 30 minutes, heat-denatured labeled probe is added, and 68 ° C / 16 hours Hybridization was carried out. After washing under the following conditions, the plate was exposed to an imaging plate (FUJI # BAS-III), and a mouse NR8-specific signal was detected with an image analyzer (FUJIX, BAS-2000II).
洗浄条件は、 （1 ) lx SSC I 0.1¾ SDS，室温で 5分、 （2 ) lx SSC / 0.1¾ SDS, 50 Cで 30分、 （3 ) 0.5x SSC / 0. 1¾ SDS， 50°Cで 30分である。  The washing conditions were (1) lx SSC I 0.1¾ SDS, 5 minutes at room temperature, (2) lx SSC / 0.1¾ SDS, 30 minutes at 50 C, (3) 0.5x SSC / 0.1¾ SDS, 50 ° C In 30 minutes.
その結果、 図 1 9に示す通り、 マウス精巣においてのみ強発現が認められた 以外、 他の臓器における同遺伝子の発現は検出されなかった。 ここで RT- PCR による解析結果とノーザン法による解析結果が異なる検出結果を示すが、 ノー ザン法の場合、 RT-PCRレベルと比較して検出感度がかなり低いため、 発現量の 低レ、 mRNAを検出することができなかったことが原因であると考えられる。しか し、 精巣における遺伝子強発現が検出されたことにより、 双方の解析結果の一 致が認められる。 また、 検出された転写産物のサイズは約 4.2 kbであった。 ノ一ザン法及び RT- PCR解析法による、各マウス臓器における遺伝子発現様態 の解析の結果においては、発現量に偏差はあったものの、特に RT- PCR法におい ては解析した全ての臓器で発現が認められるなど、 その遺伝子発現は広範な存 在分布を示した。 これは、 免疫担当組織や造血組織、 及び特定の白血病細胞株 においてのみ強い発現が認められたヒト NR8遺伝子と比較すると対照的な検出 結果であり、 その発現様態意義は大変興味深い。 このことは即ち、 マウスにお ける NR8の分子機能が、 全身性の造血機能に関与する可能性や、 或いは免疫応 答、 及び造血機能のみならず、 多岐にわたる生体の生理調節機構に関与する可 能性をも示唆しており、 つまり、 そのリガンドはホルモン様因子として機能し 得る可能性も考えられる。 As a result, as shown in Fig. 19, except for strong expression only in the mouse testis, no expression of the gene was detected in other organs. Here, the results of RT-PCR analysis are different from the results of Northern analysis, but the Northern method has significantly lower detection sensitivity compared to the RT-PCR level, resulting in lower expression levels and lower mRNA levels. Is considered to be due to the fact that could not be detected. However, the detection of strong gene expression in the testis confirms a match between the two results. The size of the transcript detected was about 4.2 kb. In the results of analysis of the gene expression pattern in each mouse organ by the Northern method and RT-PCR analysis, there was a deviation in the expression level, but especially in the RT-PCR method, expression was observed in all organs analyzed. The gene expression showed a wide distribution of presence, including the presence of This is a contrasting detection result compared to the human NR8 gene, which was strongly expressed only in immunocompetent tissues, hematopoietic tissues, and specific leukemia cell lines. This means that the mouse The molecular function of NR8 in humans may be involved in systemic hematopoietic functions, or in immune responses and hematopoietic functions, as well as in a wide variety of biological physiological regulatory mechanisms. In other words, it is possible that the ligand may function as a hormone-like factor.
[実施例 9 ] プラークスクリーニングによる NR8マウスゲノム遺伝子の単離 本発明者等は、 次に、 マウス NR8遺伝子のゲノム構造の解析を目指し、 マウ スゲノム DNAライブラリ一に対するプラークハイブリダィゼ一シヨンを試みた c ライブラリ一として Lambda F IX I Iに構築された 129SVJ株 Genomic DNA ( Str atagene#946313 )を用いた。 約 5. 0 x 105プラークのゲノムライブラリ一を展開 し、 Hybond N( + ) (Amersham #RPN303B )付電荷ナイロン膜にプロッティングした 後、 一次スクリーニングに提供した。 プローブには、 前記 7- 4)にて得られた、 5' - RACE産物の NR8 ? cDNA断片を用いた。 プローブの調製は前記 8- 2 )同様、 Me ga Prime Kitを用い [ひ-32 P]dCTPによってラジオアイソトープ標識した。 ハイ ブリダィゼ一シヨンには Express Hyb-ridization Solutionを用い、 65°C/ 30 分のプレハイプリダイゼーシヨンの後、熱変性させた標識プローブを加え、 65°C I 16時間のハイプリダイゼ一シヨンを実施した。下記の条件にて洗浄をおこな つた後、 X 線フィルム（Kodak, cat#165-1512 ) に露光し、 マウス NR8陽性ブラ —クを検出した。 [Example 9] Isolation of NR8 mouse genomic gene by plaque screening The present inventors next attempted plaque hybridization against a mouse genomic DNA library with the aim of analyzing the genomic structure of the mouse NR8 gene. As a c library, 129SVJ strain Genomic DNA (Stratagene # 946313) constructed in Lambda F IX II was used. A genomic library of about 5.0 × 10 5 plaques was developed, and plotted on a charged nylon membrane with Hybond N (+) (Amersham # RPN303B), and provided for primary screening. The probe used was the NR8 cDNA fragment of the 5'-RACE product obtained in 7-4). Preparation of probes the 8-2) Similarly, Me ga Prime Kit was used [shed - 32 P] was radioisotope-labeled by dCTP. Hybridization was performed using Express Hyb-ridization Solution, pre-hybridization at 65 ° C / 30 min, heat-denatured labeled probe was added, and hybridization at 65 ° C for 16 hours was performed. . After washing under the following conditions, exposure to X-ray film (Kodak, cat # 165-1512) was performed to detect mouse NR8 positive black.
洗浄条件は、 （1 ) lx SSC / 0. 1% SDS, 室温で 5分、 （2 ) lx SSC / 0. 1% SDS, 58°Cで 30分、 （3 ) 0.5x SSC / 0. 1% SDS, 58°Cで 30分である。  The washing conditions were (1) lx SSC / 0.1% SDS, 5 minutes at room temperature, (2) lx SSC / 0.1% SDS, 30 minutes at 58 ° C, (3) 0.5x SSC / 0.1 % SDS, 30 minutes at 58 ° C.
その結果、 陽性、 或いは疑陽性を示す、 独立した 16クローンが得られた。一 次スクリーニングによって得られた、 この 16クローンに対し、 同様に二次スク リーニングをおこなった結果、 M8陽性を示す、 独立した 9 クローンのブラ一 クの単離に成功した。 産業 _上の利用の可能性 bo As a result, 16 independent clones showing positive or false positive were obtained. Similarly, secondary screening was performed on these 16 clones obtained by the primary screening. As a result, black M9-positive 9 independent clones were successfully isolated. Industry_Possibility of use bo
本発明により新規なへモポェチン受容体蛋白質「NR8」及びそれをコ一ドする D N Aが提供された。 また、 該 D N Aが挿入されたべクタ一、 該 D N Aを保持 する形質転換体、 該形質転換体を利用した組換え蛋白質の製造方法が提供され た。 さらに、 該蛋白質に結合する天然のリガンドあるいは化合物のスクリー二 ング方法が提供された。 本発明の NR8蛋白質は、 造血作用に関連していると考 えられ、 造血作用の解析に有用である。 また、 造血関連疾患の診断や治療への 応用が期待される。 The present invention provides a novel hemopoietin receptor protein "NR8" and a DNA encoding the same. Also provided are a vector into which the DNA has been inserted, a transformant carrying the DNA, and a method for producing a recombinant protein using the transformant. Further, a method for screening a natural ligand or compound that binds to the protein was provided. The NR8 protein of the present invention is considered to be related to hematopoietic action, and is useful for analyzing hematopoietic action. It is also expected to be applied to diagnosis and treatment of hematopoietic diseases.
マウス NR8遺伝子の発現は、 マウス臓器において広範な存在分布を示したこ とから、 マウス NR8蛋白質は、 上記造血作用も含めて、 多岐にわたる生体の生 理調節機構に関与する可能性が存在する。 また、 マウス NR8蛋白質を用いれば、 まず、 マウス NR8 リガンドを単離し、 次いで、 マウス NR8 リガンドの保存構造 を利用して NR8 リガンドのヒト相同遺伝子を単離することが可能である。 具体 的には、マウス NR8リガンド cDNAの塩基配列を決定した後、 その配列上にオリ ゴヌクレオチドプライマ一を設計し、それらを用いヒト由来の cDNAライブラリ —を錶型として交叉 PCRを行なうことで、ヒト NR8 リガンド cDNAを得ることが 可能である。あるいはマウス NR8 リガンド cDNAをプローブとして、 ヒト由来の cDNAライブラリ一に対する交叉ハイプリダイゼ一シヨンを行なうことで、 ヒト NR8リガンド cDNAを得ることも可能である。 また、 マウス NR8遺伝子を利用し て、 マウス NR8 遺伝子が欠損したマウスを作成することにより、 NR8受容体蛋 白質のさらなる生体機能解析を行なうことが可能である。  Since the expression of the mouse NR8 gene showed a wide distribution in mouse organs, there is a possibility that the mouse NR8 protein is involved in a wide variety of biological physiological regulatory mechanisms including the above-mentioned hematopoietic action. When the mouse NR8 protein is used, it is possible to first isolate a mouse NR8 ligand, and then to isolate a human homologous gene of the NR8 ligand using the conserved structure of the mouse NR8 ligand. Specifically, after determining the nucleotide sequence of mouse NR8 ligand cDNA, oligonucleotide primers are designed on the sequence, and cross-PCR is performed by using them and using a human-derived cDNA library as a 錶 type. It is possible to obtain human NR8 ligand cDNA. Alternatively, human NR8 ligand cDNA can be obtained by performing cross-hybridization on a human-derived cDNA library using mouse NR8 ligand cDNA as a probe. Further, by using the mouse NR8 gene to generate a mouse lacking the mouse NR8 gene, it is possible to further analyze the biological function of the NR8 receptor protein.

Claims

ο7 請求の範囲 ο7 Claims
1 . 配列番号： 1に示す 1位のアミノ酸 M e tから 3 6 1位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のアミノ酸配列にお いて 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他のアミノ酸による 置換により修飾されたアミノ酸配列からなり、 配列番号： 1に示す 1位のアミ ノ酸 M e tから 3 6 1位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質 と機能的に同等な蛋白質。 1. A protein consisting of the amino acid sequence from amino acid 1 at position 1 to amino acid Ser at position 36 1 shown in SEQ ID NO: 1, or deletion of one or more amino acids in the amino acid sequence in the protein A protein comprising an amino acid sequence modified by addition and / or substitution with another amino acid, and comprising an amino acid sequence from amino acid Met at position 1 to amino acid Ser at position 361, as shown in SEQ ID NO: 1. Functionally equivalent protein.
2 . 配列番号： 3に示す 1位のアミノ酸 M e tから 1 4 4位のアミノ酸 L e uまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のアミノ酸配列におい て 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他のアミノ酸による置 換により修飾されたアミノ酸配列からなり、 配列番号： 3に示す 1位のアミノ 酸 M e から 1 4 4位のアミノ酸 L e uまでのアミノ酸配列からなる蛋白質と 機能的に同等な蛋白質。  2. a protein consisting of the amino acid sequence from the first amino acid Met to the 144th amino acid Leu shown in SEQ ID NO: 3, or deletion of one or more amino acids in the amino acid sequence in the protein; A protein and function comprising an amino acid sequence modified by addition and / or substitution with another amino acid, and comprising an amino acid sequence from amino acid Me at position 1 to amino acid Leu at position 144 shown in SEQ ID NO: 3 Equivalent protein.
3 . 配列番号： 5に示す 1位のアミノ酸 M e tから 2 3 7位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のアミノ酸配列にお いて 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他のアミノ酸による 置換により修飾されたアミノ酸配列からなり、 配列番号： 5に示す 1位のアミ ノ酸 M e tから 2 3 7位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質 と機能的に同等な蛋白質。  3. A protein consisting of the amino acid sequence from the first amino acid Met to the 23rd amino acid Ser shown in SEQ ID NO: 5, or deletion of one or more amino acids in the amino acid sequence in the protein A protein comprising an amino acid sequence modified by addition and / or substitution with another amino acid, and comprising an amino acid sequence from amino acid Met at position 1 to amino acid Ser at position 237 shown in SEQ ID NO: 5; Functionally equivalent protein.
4 . 配列番号： 7に示す 1位のアミノ酸 M e tから 5 3 8位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のアミノ酸配列にお いて 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他のアミノ酸による 置換により修飾されたアミノ酸配列からなり、 配列番号： 7に示す 1位のアミ ノ酸 M e tから 5 3 8位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質 と機能的に同等な蛋白質。 bo 4. A protein consisting of the amino acid sequence from the 1st amino acid Met to the 538th amino acid Ser shown in SEQ ID NO: 7, or a deletion of one or more amino acids in the amino acid sequence in the protein A protein comprising an amino acid sequence modified by addition and / or substitution with another amino acid, and comprising an amino acid sequence from the amino acid Met at position 1 to the amino acid Ser at position 538 shown in SEQ ID NO: 7. Functionally equivalent protein. bo
5. 配列番号： 19示す 1位のアミノ酸 Me tから 144位のアミノ酸 L e uまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のアミノ酸配列にお いて 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他のアミノ酸による 置換により修飾されたアミノ酸配列からなり、 配列番号： 19に示す 1位のァ ミノ酸 Me tから 144位のアミノ酸 L euまでのアミノ酸配列からなる蛋白 質と機能的に同等な蛋白質。 5. SEQ ID NO: 19: a protein consisting of the amino acid sequence from amino acid Me at position 1 to amino acid Leu at position 144 shown in SEQ ID NO: 19, or deletion or addition of one or more amino acids in the amino acid sequence in the protein And / or consists of an amino acid sequence modified by substitution with another amino acid, and is functionally equivalent to a protein consisting of the amino acid sequence from amino acid Met at position 1 to amino acid Leu at position 144 shown in SEQ ID NO: 19. Protein.
6. 配列番号： 21示す 1位のアミノ酸 Me tから 538位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質、 又は該蛋白質中のアミノ酸配列にお いて 1若しくは複数個のアミノ酸の欠失、 付加及び/又は他のアミノ酸による 置換により修飾されたアミノ酸配列からなり、 配列番号： 21に示す 1位のァ ミノ酸 Me tから 538位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白 質と機能的に同等な蛋白質。  6. SEQ ID NO: 21: a protein consisting of the amino acid sequence from the amino acid at position 1 Met to the amino acid at position 538 Ser, or deletion, addition and deletion of one or more amino acids in the amino acid sequence in the protein / Or consists of an amino acid sequence modified by substitution with another amino acid, and is functionally equivalent to a protein consisting of the amino acid sequence from amino acid Met at position 1 to amino acid Ser at position 538 shown in SEQ ID NO: 21 Protein.
7. 配列番号： 2に記載の塩基配列からなる DNAとハイブリダィズする DN Aがコードする蛋白質であって、 配列番号： 1に示す 1位のアミノ酸 Me tから 361位のアミノ酸 Se rまでのアミノ酸配列からなる蛋白質と機能的 に同等な蛋白質。  7. A protein encoded by DNA that hybridizes with DNA consisting of the nucleotide sequence of SEQ ID NO: 2, comprising an amino acid sequence from amino acid Met at position 1 to amino acid Ser at position 361 shown in SEQ ID NO: 1. A protein functionally equivalent to a protein consisting of
8. 配列番号 ： 4に記載の塩基配列からなる DNAとハイブリダィズする DNAがコードする蛋白質であって、 配列番号： 3に示す 1位のアミノ酸 Me tから 144位のアミノ酸 Leuまでのアミノ酸配列からなる蛋白質と機能的 に同等な蛋白質。  8. SEQ ID NO: a protein encoded by a DNA that hybridizes with DNA consisting of the nucleotide sequence of SEQ ID NO: 4, consisting of the amino acid sequence from the 1st amino acid Met to the 144th amino acid Leu shown in SEQ ID NO: 3 A protein that is functionally equivalent to a protein.
9. 配列番号 ： 6に記載の塩基配列からなる DNAとハイブリダィズする DNAがコードする蛋白質であって、 配列番号： 5に示す 1位のアミノ酸 Me tから 237位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質と機能的 に同等な蛋白質。  9. SEQ ID NO: a protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 6, from the amino acid sequence from the 1st amino acid Met to the 237th amino acid Ser shown in SEQ ID NO: 5 A protein that is functionally equivalent to another protein.
10. 配列番号： 8に記載の塩基配列からなる DNAとハイブリダィズす る DNAがコードする蛋白質であって、 配列番号： 7に示す 1位のアミノ酸 M e tから 538位のアミノ酸 S e rまでのアミノ酸配列からなる蛋白質と機能 的に同等な蛋白質。 10. A protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 8, wherein the amino acid at position 1 shown in SEQ ID NO: 7 is M A protein functionally equivalent to the protein consisting of the amino acid sequence from et to amino acid Ser at position 538.
1 1. 配列番号： 20に記載の塩基配列からなる D N Aとハイプリダイズ する DNAがコードする蛋白質であって、 配列番号： 19に示す 1位のアミノ 酸 Me tから 144位のアミノ酸 L e uまでのァミノ酸配列からなる蛋白質と 機能的に同等な蛋白質。  1 1. A protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 20, which comprises a sequence from amino acid Met at position 1 to amino acid Leu at position 144 shown in SEQ ID NO: 19. A protein functionally equivalent to a protein consisting of an amino acid sequence.
12. 配列番号： 22に記載の塩基配列からなる DN Aとハイブリダィズ する DNAがコードする蛋白質であって、 配列番号： 21に示す 1位のアミノ 酸 M e tから 538位のアミノ酸 Serまでのァミノ酸配列からなる蛋白質と 機能的に同等な蛋白質。  12. A protein encoded by a DNA that hybridizes to DNA consisting of the nucleotide sequence of SEQ ID NO: 22, wherein the amino acid from the 1st amino acid Met to the 538th amino acid Ser shown in SEQ ID NO: 21 A protein functionally equivalent to a protein consisting of a sequence.
13. 請求項 1〜12のいずれか 1項に記載の蛋白質と他のぺプチド又は ポリべプチドとからなる融合蛋白質。  13. A fusion protein comprising the protein according to any one of claims 1 to 12 and another peptide or polypeptide.
14. 請求項 1~13のいずれか 1項に記載の蛋白質をコードする DNA。 14. A DNA encoding the protein according to any one of claims 1 to 13.
15. 請求項 14に記載の DNAが挿入されたべクタ一。 15. A vector into which the DNA according to claim 14 has been inserted.
16. 請求項 14に記載の DN Aを発現可能に保持する形質転換体。  16. A transformant that retains the DNA of claim 14 in an expressible manner.
17. 請求項 16に記載の形質転換体を培養する工程を含む、 請求項 1〜 13のいずれか 1項に記載の蛋白質の製造方法。  17. A method for producing the protein according to any one of claims 1 to 13, comprising a step of culturing the transformant according to claim 16.
18. 請求項 1〜13に記載の蛋白質に結合する化合物をスクリーニング する方法であって、  18. A method for screening a compound that binds to the protein according to claim 1, wherein
(a)請求項 1〜 13のいずれか 1項に記載の蛋白質に被験試料を接触させる 工程、 および  (a) contacting the test sample with the protein according to any one of claims 1 to 13, and
(b) 請求項 1〜 13のいずれか 1項に記載の蛋白質に結合する活性を有する 化合物を選択する工程、 を含む方法。  (b) selecting a compound having an activity of binding to the protein according to any one of claims 1 to 13.
19. 請求項 1〜12のいずれか 1項に記載の蛋白質に対して特異的に結 合する抗体。  19. An antibody that specifically binds to the protein according to any one of claims 1 to 12.
20. 請求項 19に記載の抗体と、 請求項 1〜 13のいずれか 1項に記載 の蛋白質が含まれると予想される試料とを接触せしめ、 前記抗体と該蛋白質と の免疫複合体の生成を検出又は測定することを含んでなる該蛋白質の検出又は 測定方法。 20. The antibody according to claim 19 and the antibody according to any one of claims 1 to 13. A method for detecting or measuring said protein, comprising contacting a sample suspected of containing said protein with said sample, and detecting or measuring the production of an immune complex between said antibody and said protein.
2 1. 配列番号： 2、 4、 6、 8、 20、 および 22から 27のいずれか 一つに記載の塩基配列からなる DNAと特異的にハイプリダイズし、 少なくと も 1 5塩基の鎖長を有する DNA。  2 1. SEQ ID NO: 2, 4, 6, 8, 20, and 22 to 27, specifically hybridize with DNA consisting of the nucleotide sequence described in any one of them, and have a chain length of at least 15 bases. DNA having
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AU4392299A (en) 2000-01-10

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